Ligand (ACT-4-L) to a receptor on the surface of activated CD4+ T-cells

ABSTRACT

The invention provides ligands and fragments thereof to a receptor on the surface of activated CD4 +  T-cells. An exemplary ligand is designated ACT-4-L-h-1. Preferred fragments include purified extracellular domains of ligands. The invention also provides humanized and human antibodies to the ligand. The invention further provides methods of using the ligand and the antibodies in treatment of diseases and conditions of the immune system. The invention also provides methods of monitoring activated CD4 +  T-cells using the ligands or fragments thereof.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] Copending application U.S. Ser. No. 08/147,784, filed Nov. 3,1993, describes related subject matter and is incorporated by referencein its entirety for all purposes.

TECHNICAL FIELD

[0002] This invention relates generally to the isolation andcharacterization of a ligand (ACT-4-L) to a receptor on the surface ofactivated CD4⁺ T-cells. This invention also provides antibodies to theligand, and methods of using the ligand and the antibodies formonitoring and/or modulating immune responses.

BACKGROUND OF THE INVENTION

[0003] Immune responses are largely mediated by a diverse collection ofperipheral blood cells termed leukocytes. The leukocytes includelymphocytes, granulocytes and monocytes. Granulocytes are furthersubdivided Into neutrophils, eosinophils and basophils. Lymphocytes arefurther subdivided into T and B lymphocytes. T-lymphocytes originatefrom lymphocytic-committed stem cells of the embryo. Differentiationoccurs in the thymus and proceeds through prothymocyte, corticalthymocyte and medullary thymocyte intermediate stages, to producevarious types of mature T-cells. These subtypes include CD8⁺ T cells(also known as cytotoxic/suppressor T cells), which, when activated,have the capacity to lyse target cells, and CD4⁺ T cells (also known asT helper and T inducer cells), which, when activated, have the capacityto stimulate other immune system cell types.

[0004] Immune system responses are elicited in several differingsituations. The most frequent response is as a desirable protectionagainst infectious microorganisms. However, undesired immune responsecan occur following transplantation of foreign tissue, or in anautoimmune disease, in which one of a body's own antigens is the targetfor the immune response. Immune responses can also be initiated in vitroby mitogens or antibodies against certain receptors. In each of thesesituations, an immune response is transduced from a stimulating eventvia a complex interaction of leukocytic cell types. However, theparticipating cell types and nature of the interaction between celltypes may vary for different stimulating events. For example, immuneresponses against invading bacteria are often transduced by formation ofcomplexes between an MHC Class II receptor and a bacterial antigen,which then activate CD4⁺ T-cells. By contrast, immune responses againstviral infections are principally transduced by formation of MHC ClassI/viral antigen complexes and subsequent activation of CD8⁺ cells.

[0005] Over recent years, many leukocyte cell surface antigens have beenidentified, some of which have been shown to have a role in signaltransduction. It has been found that signals may be transduced between acell-surface receptor and either a soluble ligand or acell-surface-bound ligand. The amino acid sequences of leukocyte surfacemolecules comprise a number of characteristic recurring sequences ormotifs. These motifs are predicted to be related in evolution, havesimilar folding patterns and mediate similar types of interactions. Anumber of superfamilies, including the immunoglobulin and nerve growthfactor receptor superfamilies, have been described. Members of the nervegrowth factor receptor family include NGFR, found on neural cells; theB-cell antigen CD40; the rat OX-40 antigen, found on activated CD4⁺cells (Mallet et al., EMBO J. 9:1063-1068 (1990) (hereby incorporated byreference for all purposes); two receptors for tumor necrosis factor(TNF), LTNFR-1 and TNFR-II, found on a variety of cell types; 4-1BBfound on T-cells; SFV-T2, an open reading frame in Shope fibroma virus;and possibly fas, CD27 and CD30. See generally Mallet & Barclay,Immunology Today 12:220-222 (1990) (hereby incorporated by reference forall purposes).

[0006] The identification of cell-surface receptors has suggested newagents for suppressing undesirable immune responses such as transplantrejection, autoimmune disease and inflammation. Agents, particularlyantibodies, that block receptors of immune cells from binding to solublemolecules or cell-bound receptors can impair immune responses. Ideally,an agent should block only undesired immune responses (e.g., transplantrejection) while leaving a residual capacity to effect desirableresponses (e.g., responsive to pathogenic microorganisms). Theimmunosuppressive action of some agents, for example, antibodies againstthe CD3 receptor and the IL-2 receptor have already been tested inclinical trials. Although some trials have shown encouraging results,significant problems remain. First, a patient may develop an immuneresponse toward the blocking agent preventing continuedimmunosuppressive effects unless different agents are available. Second,cells expressing the target antigen may be able to adapt to the presenceof the blocking agent by ceasing to express the antigen, while retainingimmune functions. In this situation, continued treatment with a singleimmunosuppressive agent is ineffective. Third, many targets fortherapeutic agents are located on more than one leukocyte subtype, withthe result that it is generally not possible to selectively block oreliminate the response of only specific cellular subtypes and therebyleave unimpaired a residual immune capacity for combating infectiousmicroorganisms.

[0007] Based on the foregoing it is apparent that a need exists foradditional and improved agents capable of suppressing immune responses,particularly agents capable of selective suppression. The presentinvention fulfills these and other needs, in part, by providing a ligand(ACT-4-L) to a receptor localized on activated human CD4⁺ T-lymphocytes.

SUMMARY OF THE INVENTION

[0008] The invention provides purified ACT-4-L ligand polypeptides. Thepolypeptides have a segment between 5-160 contiguous amino acids fromthe amino acid of an exemplified ACT-4-L ligand designated ACT-4-L-h-1.The polypeptides usually exhibit at least 80% sequence identity to theACT-4-h-L-1 sequence and often share an antigenic determinant in commonwith the ACT-4-L-h-1 ligand. Usually, the polypeptides comprise anextracellular domain.

[0009] The invention also provides purified extracellular domains ofACT-4-L ligands. These domains comprise at least five contiguous aminoacids from the full-length ACT-4-L-h-1 extracellular domain. Some ofthese extracellular domains are full-length. Other extracellular domainsare fragments of full-length domains. Some extracellular domainsspecifically bind to the ACT-4-L-h-1 ligand. Other extracellular domainsspecifically bind to an exemplified receptor of ACT-4-L-h-1, thereceptor being designated ACT-4-h-1. Some extracellular domains consistessentially of a domain possessing a particular functional property, forexample, the capacity to specifically bind to the ACT-4-h-1 receptor.Some extracellular domains inhibit in vitro activation of CD4⁺ T-cellsexpressing the ACT-4-h-1 receptor on their surface. Other extracellulardomains stimulate in vitro activation of such T-cells. Any of the aboveextracellular domains may further comprise a linked second polypeptidesuch as the constant region of an immunoglobulin heavy chain.

[0010] The invention also provides an ACT-4 receptor polypeptideconsisting essentially of a domain that specifically binds to theACT-4-L-h-1 ligand.

[0011] The invention further provides antibodies that specifically bindto ACT-4-L-h-1, preferably to an extracellular domain thereof. Preferredantibodies are humanized antibodies and human antibodies. The antibodieshave a variety of binding specificities. For example, some humanizedantibodies specifically bind to the ACT-4-L-h-1 ligand on the surface ofa B-cell so as to inhibit activation of the B cell. Other antibodiesstimulate activation of B-cells. Other antibodies specifically bind tothe ACT-4-L-h-1 ligand on the surface of a B-cell so as to inhibit thecapacity of the B-cell to activate CD4⁺ T-cells.

[0012] In another aspect, the invention provides pharmaceuticalcompositions. The pharmaceutical compositions comprise apharmaceutically active carrier and an agent that specifically binds toan extracellular domain of the ACT-4-L-h-1 ligand.

[0013] The invention further provides methods of suppressing an immuneresponse in a patient having an immune disease or condition. The methodscomprise administering an effective amount of a pharmaceuticalcomposition comprising a pharmaceutically active carrier and an agentthat specifically binds to the ACT-4-L-h-1 ligand. Preferred agents aremonoclonal antibodies, ACT-4-L ligand polypeptides and ACT-4 receptorpolypeptides. The invention provides other methods of suppressing animmune response in which the agent competes with the ACT-4-L-h-1 ligandfor specific binding to the ACT-4-h-1 receptor.

[0014] The invention also provides methods of screening forimmunosuppressive agents. The methods comprise contacting an ACT-4-L-h-1ligand polypeptide with a potential immunosuppressive agent. Specificbinding between the ACT-4-L-h-1 ligand polypeptide and the agent isdetected. The specific binding is indicative of immunosuppressiveactivity.

[0015] The invention also provides methods of monitoring activated CD4⁺T-cells. The methods comprise contacting a tissue sample from a patientwith an ACT-4-L ligand polypeptide that specifically binds to anextracellular domain of the ACT-4-h-1 receptor. Specific binding betweenthe ACT-4-L ligand polypeptide and the tissue sample is detected toindicate the presence of the activated CD4⁺ T-cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1: Two-color staining of peripheral blood lymphocytes toanalyze expression of ACT-4-h-1 on different cell types.

[0017]FIG. 2: Kinetics of ACT-4-h-1 expression on alloantigen-activatedCD4⁺ T-cells. MCF=Mean channel fluorescence.

[0018]FIG. 3: Kinetics of ACT-4-h-1 expression ontetanus-toxoid-activated CD4⁺ T-cells.

[0019]FIG. 4: Kinetics of ACT-4-h-1 expression on PHA-activated CD4⁺T-cells.

[0020]FIG. 5: cDNA (upper) and deduced amino acid sequence (lower) ofACT-4-h-1. The Figure indicates the locations of an N-terminal signalsequence, two possible signal cleavage sites (vertical arrows), twoglycosylation sites (gly), a transmembrane domain (TM), a stop codon anda poly-A signal sequence.

[0021]FIG. 6: Construction of expression vector for production of stabletransfectants expressing ACT-4-h-1.

[0022]FIG. 7: FACS analysis showing expression of ACT-4-h-1 on stabletransfectants of COS-7, Jurkat and SP2/O cell lines.

[0023]FIG. 8: Fusion of an ACT-4-h-1 extracellular domain with animmunoglobulin heavy chain constant region to form a recombinantglobulin.

[0024]FIG. 9: Schematic topographical representation of recombinantglobulin formed from fusion of an ACT-4-h-1 extracellular domain with animmunoglobulin heavy chain constant region to form a recombinantglobulin.

[0025]FIG. 10: cDNA sequence and predicted amino acid sequence ofACT-4-L-h-1. Boxed regions designate a transmembrane domain, fourglycosylation sites and a poly-A signal.

DEFINITIONB

[0026] Abbreviations for the twenty naturally occurring amino acidsfollow conventional usage (Immunology—A Synthesis, (E. S. Golub & D. R.Gren, eds., Sinauer Associates, Sunderland, Mass., 2nd ed., 1991)(hereby incorporated by reference for all purposes). Stereoisomers(e.g., D-amino acids) of the twenty conventional amino acids, unnaturalamino acids such as α,α-disubstituted amino acids, N-alkyl amino acids,lactic acid, and other unconventional amino acids may also be suitablecomponents for polypeptides of the present invention. Examples ofunconventional amino acids include: 4-hydroxyproline,γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine,O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine,5-hydroxylysine, ω-N-methylarginine, and other similar amino acids andimino acids (e.g., 4-hydroxyproline). In the polypeptide notation usedherein, the left-hand direction is the amino terminal direction and theright-hand direction is the carboxy-terminal direction, in accordancewith standard usage and convention. Similarly, unless specifiedotherwise, the lefthand end of single-stranded polynucleotide sequencesis the 5′ end; the lefthand direction of double-stranded polynucleotidesequences is referred to as the 5′ direction. The direction of 5′ to 3′addition of nascent RNA transcripts is referred to as the transcriptiondirection; sequence regions on the DNA strand having the same sequenceas the RNA and which are 5′ to the 5′ end of the RNA transcript arereferred to as “upstream sequences”; sequence regions on the DNA strandhaving the same sequence as the RNA and which are 3′ to the 3′ end ofthe RNA transcript are referred to as “downstream sequences”.

[0027] The phrase “polynucleotide sequence” refers to a single ordouble-stranded polymer of deoxyribonucleotide or ribonucleotide basesread from the 5′ to the 3′ end. It includes self-replicating plasmids,infectious polymers of DNA or RNA and non-functional DNA or RNA.

[0028] The following terms are used to describe the sequencerelationships between two or more polynucleotides: “reference sequence”,“comparison window”, “sequence identity”, “percentage of sequenceidentity”, and “substantial identity”. A “reference sequence” is adefined sequence used as a basis for a sequence comparison; a referencesequence may be a subset of a larger sequence, for example, as a segmentof a full-length cDNA or gene sequence given in a sequence listing, suchas a polynucleotide sequence shown in FIG. 5 or FIG. 10, or may comprisea complete cDNA or gene sequence. Generally, a reference sequence is atleast 20 nucleotides in length, frequently at least 25 nucleotides inlength, and often at least 50 nucleotides in length. Since twopolynucleotides may each (1) comprise a sequence (i.e., a portion of thecomplete polynucleotide sequence) that is similar between the twopolynucleotides, and (2) may further comprise a sequence that isdivergent between the two polynucleotides, sequence comparisons betweentwo (or more) polynucleotides are typically performed by comparingsequences of the two polynucleotides over a “comparison window” toidentify and compare local regions of sequence similarity. A “comparisonwindow”, as used herein, refers to a conceptual segment of at least 20contiguous nucleotide positions wherein a polynucleotide sequence may becompared to a reference sequence of at least 20 contiguous nucleotidesand wherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith & Waterman, Appl.Math. 2:482 (1981), by the homology alignment algorithm of Needleman &Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity methodof Pearson & Lipman, Proc. Natl. Acad. Sci. (USA) 85:2444 (1988), bycomputerized implementations of these algorithms (FASTDB(Intelligenetics), BLAST (National Center for Biomedical Information) orGAP, BESTFIT, FASTA, and TFASTA (Wisconsin Genetics Software PackageRelease 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.)),or by inspection, and the best alignment (i.e., resulting in the highestpercentage of sequence similarity over the comparison window) generatedby the various methods is selected. The term “sequence identity” meansthat two polynucleotide sequences are identical (i.e., on anucleotide-by-nucleotide basis) over the window of comparison. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. The terms “substantial identity” as used herein denotes acharacteristic of a polynucleotide sequence, wherein the polynucleotidecomprises a sequence that has at least 70, 80 or 85 percent sequenceidentity, preferably at least 90 to 95 percent sequence identity, moreusually at least 99 percent sequence identity as compared to a referencesequence over a comparison window of at least 20 nucleotide positions,frequently over a window of at least 25-50 nucleotides, wherein thepercentage of sequence identity is calculated by comparing the referencesequence to the polynucleotide sequence which may include deletions oradditions which total 20 percent or less of the reference sequence overthe window of comparison. The reference sequence may be a subset of alarger sequence, for example, as a segment of the full-length ACT-4-h-1sequence shown in FIG. 5 or a segment of the full-length ACT-4-L-h-1sequence shown in FIG. 10.

[0029] As applied to polypeptides, the term “substantial identity” meansthat two peptide sequences, when optimally aligned, such as by theprograms BLAZE (Intelligenetics) GAP or BESTFIT using default gapweights, share at least 70 percent or 80 percent sequence identity,preferably at least 90 percent sequence identity, more preferably atleast 95 percent sequence identity or more (e.g., 99 percent sequenceidentity). Preferably, residue positions which are not identical differby conservative amino acid substitutions. Conservative amino acidsubstitutions refer to the interchangeability of residues having similarside chains. For example, a group of amino acids having aliphatic sidechains is glycine, alanine, valine, leucine, and isoleucine; a group ofamino acids having aliphatic-hydroxyl side chains is serine andthreonine; a group of amino acids having amide-containing side chains isasparagine and glutamine; a group of amino acids having aromatic sidechains is phenylalanine, tyrosine, and tryptophan; a group of aminoacids having basic side chains is lysine, arginine, and histidine; and agroup of amino acids having sulfur-containing side chains is cysteineand methionine. Preferred conservative amino acids substitution groupsare: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine.

[0030] The term “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition will comprise more than about 80 to 90 percent of allmacromolecular species present in the composition. Most preferably, theobject species is purified to essential homogeneity (contaminant speciescannot be detected in the composition by conventional detection methods)wherein the composition consists essentially of a single macromolecularspecies.

[0031] The term “naturally-occurring” as used herein as applied to anobject refers to the fact that an object can be found in nature. Forexample, a polypeptide or polynucleotide sequence that is present in anorganism (including viruses) that can be isolated from a source innature and which has not been intentionally modified by man in thelaboratory is naturally-occurring.

[0032] The term “epitope” includes any protein determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. Epitopicdeterminants usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics.

[0033] Specific binding exists when the dissociation constant for adimeric complex is ≦1 μM, preferably ≦100 nM and most preferably ≦1 nM.

[0034] The term “higher cognate variants” as used herein refers to agene sequence that is evolutionarily and functionally related betweenhumans and higher mammalian species, such as primates, porcines andbovines. The term does not include gene sequences from rodents, such asrats. Thus, the cognate primate gene to the ACT-4-h-1 gene is theprimate gene which encodes an expressed protein which has the greatestdegree of sequence identity to the ACT-4-h-1 receptor protein and whichexhibits an expression pattern similar to that of the ACT-4-h-1 protein(i.e., expressed on activated CD4⁺ cells). Similarly, the cognateprimate gene to the ACT-4-L-h-1 gene is the gene whose expressed proteinshows greatest sequence identity to the ACT-4-L-h-1 ligand protein andwhich exhibits a similar expression pattern (i.e., expressed onactivated B-cells).

[0035] A population of cells is substantially enriched in a selectedcell type when that cell type constitutes at least 30, 50 or 70% of thepopulation.

[0036] The term “patient” includes human and veterinary subjects.

[0037] A test substance competes with a reference for specific bindingto an antigen when an excess of the test substance substantiallyinhibits binding of the reference in a competition assay. Numerous typesof competition assay including radioimmunoassay and ELISA are available.See Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor(1988). Substantially inhibits means that the test substance reducesspecific binding of the reference usually by at least 10%, 25%, 50%,75%, or 90%. Test substances identified by a competition assay includethose binding to the same epitope as the reference and those binding toan adjacent epitope sufficiently proximal to that of the epitope boundby the reference antibody for steric hindrance to occur.

DETAILED DESCRIPTION

[0038] I. ACT-4 Receptor Polypeptides

[0039] According to one embodiment of the invention, receptors on thesurface of activated CD4⁺ T-cells (referred to as ACT-4 receptors) andfragments thereof are provided. The term ACT-4 receptor polypeptide isused generically to encompass full-length proteins and fragmentsthereof. The term ACT-4 receptor is usually reserved for full-lengthproteins. The amino acid sequence of the first ACT-4 receptor to becharacterized [hereinafter ACT-4-h-1] is shown in FIG. 5. The suffix -hdesignates human origin and the suffix -1 indicates that ACT-4-h-1 isthe first ACT-4 receptor to be characterized. The term ACT-4 receptorrefers not only to the protein having the sequence shown in FIG. 5, butalso to other proteins that represent allelic, nonallelic, and highercognate variants of ACT-4-h-1, and natural or induced mutants of any ofthese. Usually, ACT-4 receptor polypeptides will also show substantialsequence identity with the ACT-4-h-1 sequence. Typically, an ACT-4receptor polypeptide will contain at least 4 and more commonly 5, 6, 7,10 or 20, 50 or more contiguous amino acids from the ACT-4-h-1 sequence.It is well known in the art that functional domains, such as bindingdomains or epitopes can be formed from as few as four amino acidsresidues.

[0040] ACT-4 receptor polypeptides will typically exhibit substantialamino acid sequence identity with the amino acid sequence of ACT-4-h-1,and be encoded by nucleotide sequences that exhibit substantial sequenceidentity with the nucleotide sequence encoding ACT-4-h-1 shown in FIG.5. The nucleotides encoding ACT-4 receptor proteins will also typicallyhybridize to the ACT-4-h-1 sequence under stringent conditions. However,these nucleotides will not usually hybridize under stringent conditionsto the nucleic acid encoding OX-40 receptor, as described by Mallet etal., EMBO J. 9:1063-68 (1990) (hereby incorporated by reference for allpurposes) (See particularly FIG. 2A of the Mallet et al. reference).Stringent conditions are sequence dependent and will be different indifferent circumstances. Generally, stringent conditions are selected tobe about 5° C. lower than the thermal melting point (Tm) for thespecific sequence at a defined ionic strength and pH. The Tm is thetemperature (under defined ionic strength and Ph) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Typically,stringent conditions will be those in which the salt concentration is atleast about 0.02 molar at Ph 7 and the temperature is at least about 60°C. As other factors may significantly affect the stringency ofhybridization, including, among others, base composition and size of thecomplementary strands, the presence of organic solvents and the extentof base mismatching, the combination of parameters is more importantthan the absolute measure of any one.

[0041] Usually, ACT-4 receptor polypeptides will share at least oneantigenic determinant in common with ACT-4-h-1 but will not bespecifically reactive with antibodies against the rat OX-40 polypeptide.The existence of a common antigenic determinant is evidenced bycross-reactivity of the variant protein with any antibody preparedagainst ACT-4-h-1 (see Section IV). Cross-reactivity is often testedusing polyclonal sera against ACT-4-h-1, but can also be tested usingone or more monoclonal antibodies against ACT-4-h-1, such as theantibody designated L106.

[0042] Often ACT-4 receptor polypeptides will contain modifiedpolypeptide backbones. Modifications include chemical derivatizations ofpolypeptides, such as acetylations, carboxylations and the like. Theyalso include glycosylation modifications (N- and O-linked) andprocessing variants of a typical polypeptide. These processing stepsspecifically include enzymatic modifications, such as ubiquitinizationand phosphorylation. See, e.g., Hershko & Ciechanover, Ann. Rev. Bioch.51:335-364 (1982). The ACT-4-h-1 protein, for example, is heavilymodified in that the observed molecular weight is about 50 kDa, whereasthe predicted molecular weight based on amino acid sequence is only 27kDa. Two putative glycosylation sites have been identified in itsextracellular domain.

[0043] ACT-4 receptors likely share some or all of the topologicalfeatures found for ACT-4-h-1. The amino acid sequence for ACT-4-h-1contains a 22 or 24 amino acid putative N-terminal signal sequence. The24 amino acid sequence is more probably based on the criteria of vonHeijne, Nucleic Acids Res. 14:4683-4690 (1986) (incorporated byreference for all purposes). The ACT-4-h-1 receptor contains a singleadditional hydrophobic stretch of 27 amino acids spanning residues213-240. The hydrophobic stretch probably corresponds to a transmembranedomain and its existence is consistent with ACT-4-h-1 being a type Iintegral membrane protein (i.e., having a single transmembrane domainwith the N-terminal domain comprising the extracellular region and theC-terminus comprising the intracellular region). The 189 or 191 aminoacids (depending on the exact location of the signal cleavage site) ofACT-4-h-1 amino-proximal to the transmembrane segment are designated theextracellular domain, while the 37 amino acids carboxy-proximal to thetransmembrane segment are designated the intracellular domain. From theamino-terminus, the extracellular domain has an NH₂-terminal hydrophobicputative signal sequence, and three intrachain loops formed by disulfidebonding between paired cysteine residues.

[0044] The topological arrangement of ACT-4 receptor polypeptides issimilar to that of other members of the nerve growth factor receptorfamily, particularly to the rat OX-40 receptor. However, the othermembers show some divergence in the number of extracellular disulfideloops and glycosylation sites and in the size of the intracellulardomain. See Mallet & Barclay, supra.

[0045] Although not all of the domains discussed above are necessarilypresent in all ACT-4 receptor polypeptides, an extracellular domain isexpected to be present in most. Indeed, in some ACT-4 receptorpolypeptides, it is possible that only an extracellular domain ispresent, and the natural state of such proteins is not as cell-surfacebound proteins, but as soluble proteins, for example, dispersed in anextracellular body fluid. The existence of soluble variant forms hasbeen observed for other cell surface receptors, including one member ofthe nerve growth factor receptor family, SFV-T2. See Mallet & Barclay,supra.

[0046] Besides substantially full-length polypeptides, the presentinvention provides for biologically active fragments of thepolypeptides. Significant biological activities include receptorbinding, antibody binding (e.g., the fragment competes with an intactACT-4 receptor for specific binding to an antibody), immunogenicity(i.e., possession of epitopes that stimulate B or T cell responsesagainst the fragment), and agonism or antagonism of the binding of anACT-4 receptor polypeptide to its ligands. A segment of an ACT-4receptor protein or a domain thereof will ordinarily comprise at leastabout 5, 7, 9, 11, 13, 16, 20, 40, or 100 contiguous amino acids.

[0047] Segments of ACT-4 receptor polypeptides are often terminated nearboundaries of functional or structural domains. Such segments consistessentially of the amino acids responsible for a particular functionalor structural property. Structural and functional domains are identifiedby comparison of nucleotide and/or amino acid sequence data such as isshown in FIG. 5 to public or proprietary sequence databases. Preferably,computerized comparison methods are used to identify sequence motifs orpredicted protein conformation domains that occur in other proteins ofknown structure and/or function. Structural domains include anintracellular domain, transmembrane domain, and extracellular domain,which is in turn contains three disulfide-bonded loops. Functionaldomains include an extracellular binding domain through which the ACT-4receptor polypeptide interacts with external soluble molecules or othercell-bound ligands and an intracellular signal-transducing domain.

[0048] Some fragments will contain only extracellular domains, such asone or more disulfide-bonded loops. Such fragments will often retain thebinding specificity of an intact ACT-4 receptor polypeptide, but will besoluble rather than membrane bound. Such fragments are useful ascompetitive inhibitors of ACT-4 receptor binding.

[0049] ACT-4 receptors are further identified by their status as membersof the nerve growth factor receptor family. The amino acid sequence ofACT-4-h-1 is at least 20% identical to NGF-R, TNF-R, CD40, 4-1BB, andfas/APO1. ACT-4-h-1 exhibits 62% amino acid sequence identity with therat OX-40 gene, which is also characterized by selective expression onactivated CD4⁺ cells.

[0050] ACT-4 receptors are also identified by a characteristic cellulardistribution. Most notably, ACT-4 receptors are usually easily detectedon activated CD4⁺ T cells (percent cells expressing usually greater thanabout 25 or 50% and often about 80%; mean channel fluorescence usuallygreater than about 10 and often about 20-25, on a Coulter Profile FlowCytometer after immunofluorescence staining). ACT-4 receptors areusually substantially absent on resting T-cells, B-cells (unlessactivated with PMA), NK cells, and monocytes (unless activated withPMA). Substantially absent means that the percentage of cells expressingACT-4 is usually less than about 5%, and more usually less than about2%, and that the mean channel is usually less than about 4, and moreusually less than about 2, measured on a Coulter Profile Flow Cytometer,after immunofluorescence staining of the cells. (See Example 2) ACT-4receptors are usually expressed at low levels on activated CD8⁺ cells(percent cells expressing about 4-10%; mean channel fluorescence about2-4 on a Coulter Profile Flow Cytometer after immunofluorescencestaining). The low level of expression observed on CD8⁺ cells suggeststhat expression is confined to a subpopulation of CD8⁺ cells. Theexpression of ACT-4 receptors on the surface of activated CD4⁺ cells hasbeen observed for several different mechanisms of activation, includingalloantigenic, tetanus toxoid or mitogenic (e.g., PHA) stimuli.Expression peaks after about 7 days of allogantigenic or tetanus toxoidstimulation and after about three days of PHA stimulation. These dataindicate that ACT-4 receptors should be classified as early activationantigens that are substantially absent on resting cells. The observationthat ACT-4 receptors are preferentially expressed on activated CD4⁺cells and are expressed to a much lesser extent on activated CD8⁺ cells,but are substantially absent on most or all other subtypes of lymphoidcells (except in response to highly nonphysiological stimuli such asPMA) contrasts with the cell type specificity of other activationantigens found on human leukocytes.

[0051] The expression of ACT-4 receptors on the surface of activatedCD4⁺ T cells suggests that the receptor has a role in activation ofthese cells. Such a role is consistent with that of some other membersof the nerve growth factor receptor family. For example, CD40 stimulatesthe G1-S phase transition in B lymphocytes, and nerve growth factorreceptor transduces a signal from the cytokine nerve growth factor.,which results in neuronal differentiation and survival (Barde, Neuron2:1525-1534 (1989)) (incorporated by reference for all purposes).However, other roles for ACT-4 receptors can also be envisaged, forexample, interaction with other lymphoid cell types. The existence ofsuch roles is consistent with the diverse functions of other nervegrowth factor receptor family members, such as tumor necrosis factor,whose interaction with tumor necrosis factor receptor can result ininflammation or tumor cell death.

[0052] Fragments or analogs comprising substantially one or morefunctional domain (e.g., an extracellular domain) of ACT-4 receptors canbe fused to heterologous polypeptide sequences, such that the resultantfusion protein exhibits the functional property(ies) conferred by theACT-4 receptor fragment and/or the fusion partner. The orientation ofthe ACT-4 receptor fragment relative to the fusion partner will dependon experimental considerations such as ease of construction, stabilityto proteolysis, thermal stability, immunological reactivity, amino- orcarboxyl-terminal residue modification, and so forth. Potential fusionpartners include chromogenic enzymes such as β-galactosidase, protein Aor G, a FLAG protein such as described by Blanar & Rutter, Science256:1014-1018 (1992), toxins (e.g., diphtheria toxin, Psuedonomasectotoxin A, ricin toxin or phospholipase C) and immunoglobulincomponents.

[0053] Recombinant globulins (Rg) formed by fusion of ACT-4 receptorfragments and immunoglobulin components often have most or all of thephysiological properties associated with the constant region of theparticular immunoglobulin class used. For example, the recombinantglobulins may be capable of fixing complement, mediating antibodydependent cell toxicity, stimulating B cells, or traversing blood vesselwalls and entering the interstitial space. The recombinant globulins areusually formed by fusing the C-terminus of an ACT-4 receptorextracellular domain to the N-terminus of the constant region domain ofa heavy chain immunoglobulin, thereby simulating the conformation of anauthentic immunoglobulin chain. The immunoglobulin chain is preferablyof human origin, particularly if the recombinant globulin is intendedfor therapeutic use. Recombinant globulins are usually soluble and havea number of advantageous properties relative to unmodified ACT-4receptors. These properties include prolonged serum half-life, thecapacity to lyse target cells for which an ACT-4 receptor has affinity,by effector functions, and the capacity to bind molecules such asprotein A and G, which can be used to immobilize the recombinantglobulin in binding analyses.

[0054] II. Ligands to ACT-4

[0055] The invention also provides ligands that specifically bind to anACT-4 receptor polypeptide and that are capable of forming a complexwith such a polypeptide, at least in part, by noncovalent binding. Theterm ACT-4-L ligand polypeptide is used generically to encompassfull-length proteins and fragments thereof. This term does not usuallyinclude antibodies to ACT-4 receptor polypeptides. The term ACT-4 ligandis usually used to refer to a full-length protein. Ligands can benaturally-occurring or synthetic molecules, and can be in soluble formor anchored to the surface of a cell. Multiple different ligands maybind the same ACT-4 receptor. Conversely, one ligand may bind to morethan one ACT-4 receptor. Usually, binding of a ligand to an ACT-4receptor will initiate a signal that alters the physical and/orfunctional phenotype of a cell bearing the ACT-4 receptor and/or a cellbearing the ACT-4 ligand. Antibodies against either ACT-4 or its ligandscan have the capacity to block or stimulate signal transduction. Itwill, of course, be recognized that the designation of ACT-4 as areceptor and its specific binding partner(s) as ligand(s) is somewhatarbitrary and might, in some circumstances, be reversed.

[0056] Source materials for supplying ACT-4-L ligand polypeptides areidentified by screening different cell types, particularly lymphoid andhematopoietic cells, bodily fluids and tissue extracts, with labelledACT-4 receptor polypeptides, preferably in acqueous-soluble form, as aprobe. Activated B cells or B cell lines may be suitable (see Example8). HTLV-I infected T-cells are also suitable. Often, the ACT-4 receptoror a binding fragment thereof is fused or otherwise linked to a secondprotein for purposes of screening. Particularly suitable are recombinantglobulins formed by fusing the extracellular portion of ACT-4-h-1 to theconstant region of an immunoglobulin heavy chain.

[0057] ACT-4-L ligand polypeptides are purified from cells or otherbiological materials identified by this screening method usingtechniques of classical protein chemistry. Such techniques includeselective precipitation with such substances as ammonium sulfate, columnchromatography, immunopurification methods, and others. See, e.g., R.Scopes, Protein Purification: Principles and Practice (Springer-Verlag,NY, 1982) (incorporated by reference for all purposes). Usually,purification procedures will include an affinity chromatography step inwhich an ACT-4 receptor polypeptide or a binding fragment thereof isused as the immobilized reagent. ACT-4-constant regions can beconveniently immobilized by binding of the constant region moiety toprotein A or G. ACT-4-L ligand polypeptides can also be purified usinganti-idiotypic antibodies to ACT-4 receptors as the affinity reagent.

[0058] To determine the amino acid sequence or to obtain polypeptidefragments of a ligand, the ligand can be digested with trypsin. Peptidefragments can be separated by reverse-phase high-performance liquidchromatography (HPLC), and analyzed by gas-phase sequencing. Othersequencing methods known in the art may also be used. The sequence datacan be used to design degenerate probes for isolation of cDNA or genomicclones encoding ACT-4-L ligand polypeptides.

[0059] Alternatively, cDNA clones encoding ACT-4-L ligand polypeptidescan be obtained by expression cloning. In this approach, a cDNA libraryis prepared from cells expressing an ACT-4-L ligand polypeptides(identified as discussed, supra). The library is expressed inappropriate cells (e.g., COS-7), and clones bearing the ACT-4-L ligandpolypeptide are identified by screening with labelled ACT-4 or bindingfragment thereof, optionally fused to a constant domain of animmunoglobulin heavy chain.

[0060] The cDNA sequence and predicted amino acid sequence of the firstligand to an ACT-4 receptor polypeptide to be characterized are shown inFIG. 10. This ligand is designated ACT-4-L-h-1 with the suffix hdesignating human origin, and the suffix 1 indicating that this is thefirst ligand to be characterized. The coding portion of the cDNAsequence of ACT-4-L-h-1 is identical or nearly identical to that of apolypeptide termed gp34 or TA34. See Miura et al., Mol. Cell. Biol.11:1313-1325 (1991) (incorporated by reference in its entirety for allpurposes). The invention also includes ligands representing allelic,nonallelic, splice and higher cognate variants of ACT-4-L-h-1, andnatural or induced mutants of any of these. Such variants will typicallyshow substantial sequence identity with the ACT-4-L-h-1 sequence, andcontain at least 4 and more commonly 5, 6, 7, 10 or 20, 50 or morecontiguous amino acids from the ACT-4-L-h-1 sequence. Such variants willalso typically be encoded by nucleotide sequences that exhibitsubstantial sequence identity with the nucleotide sequence encodingACT-4-L-h-1 shown in FIG. 10. The nucleotides encoding such variantswill also typically hybridize to an ACT-4-L-h-1 DNA sequence understringent conditions. However, some such nucleotides will not hybridizeunder stringent conditions to DNA sequences encoding lower cognatevariants (e.g. rat) of ACT-4-L-h-1. Many variants of ACT-4-L-h-1 willshare at least one antigenic determinant with an ACT-4-L-h-1 ligandpolypeptide as evidenced by crossreactivity with monoclonal orpolyclonal antibodies against the same. However, some such variants willnot crossreact with sera against lower cognate variants of ACT-4-L-h-1.

[0061] Although many ACT-4-L ligand polypeptides will show similarity toACT-4-L-h-1 in at least one of the respects discussed above, it isentirely possible that other families of ligands exists to the ACT-4receptor that show nothing in common with ACT-4-L-h-1 except for thecapacity to specifically bind to an ACT-4 receptor. Such families ofligands are expressly included in the invention.

[0062] Often ACT-4-L ligand polypeptides will contain modifications oftheir backbones of the types discussed in Section I, supra. TheACT-4-L-h-1 polypeptide, for example, is heavily modified in that theobserved molecular weight is about 34 kDa, whereas the predictedmolecular weight based on amino acid sequence is only 21 kDa. Fourputative N-linked glycosylation sites have been identified in theextracellular domain.

[0063] Many ACT-4-L ligand polypeptides likely share some or all of thetopological features found for ACT-4-L-h-1. The ACT-4-L-h-1 amino acidsequence contains a putative N-terminal intracellular domain (aa 1-23),a putative hydrophobic transmembrane domain (aa 24-50) and a putativeextracellular C-terminal domain (aa 51-183). This structural arrangementis consistent with ACT-4-L-h-1 being a type II integral membrane protein(i.e., having a single transmembrane domain with the C-terminal domaincomprising the extracellular region and the N-terminus comprising theintracellular region).

[0064] ACT-4-L ligand polypeptides may also share some of the structuraland/or functional characteristics of ligands that binds to other membersof the nerve growth factor receptor superfamily. Such ligands includeTNF-α, TNF-β, CO40-L, CD27-L, CD30-L. Although ACT-4-L-h-1 shows onlyweak primary amino acid sequence identity with TNF-α and even lesssimilarity with other superfamily ligands, a greater similarity betweenall of these ligands is apparent in their predicted capacity to formhigher order structures. The extracellular domains of known superfamilyligands consist of about 150 amino acids and form several β-pleatedsheets, which assemble into a slitted cylindrical structure (termed a“jelly roll” by Bazan et al., Current Biology 3:603-606 (1993))(incorporated by reference for all purposes). The extracellular domainof ACT-4-L-h-1 consists of 133 amino acids, and the predicted foldingpattern is consistent with the formation of β-pleated sheets and a“jelly roll.” Notably, all of the superfamily ligands except TNF-β alsoexist, in part, as type II integral membrane cell surface proteins.Superfamily ligands also exists as soluble proteins suggesting that suchforms exist for ACT-4-L ligand polypeptides. The C-terminalextracellular domain of ACT-4-L-h-1 shows some sequence similarity withvarious dehydrogenases. Thus, some ACT-4-L ligand polypeptide maypossess a dehydrogenase activity, which may play a role in intercellularsignalling.

[0065] Besides substantially full-length polypeptides, the presentinvention provides for biologically active fragments of full-lengthACT-4-L ligand polypeptides. Significant biological activities includebinding to an ACT-4 receptor such as ACT-4-h-1, binding to a secondACT-4-L ligand polypeptide, antibody binding (e.g., the fragmentcompetes with an intact ACT-4-L-h-1 ligand polypeptide for specificbinding to an antibody), immunogenicity (i.e., possession of epitopesthat stimulate B or T cell responses against the fragment), and agonismor antagonism of the binding of a second ACT-4-L ligand polypeptide toan ACT-4 receptor polypeptide, such as ACT-4-h-1. A segment of afull-length ACT-4-L ligand polypeptide will ordinarily comprise at least5 contiguous amino acids, but not more than 160 contiguous amino acidsfrom the amino acid sequence shown in FIG. 10. Often segments containabout 10, 20, 50, 75, 100 or 133 amino acids, and not more than 150contiguous amino acids, from the sequence shown in FIG. 10.

[0066] Some fragments will contain only extracellular domains. Suchfragments contain the full-length domains of naturally occurring ACT-4-Lligand polypeptides. other fragments contain components thereof. Suchfragments will often retain the binding specificity of an intact ACT-4-Lligand polypeptide, but will be soluble rather than membrane bound. Suchfragments are useful as competitive inhibitors of ACT-4-L ligandpolypeptide binding to a receptor.

[0067] Fragments of full-length ACT-4-L ligand polypeptides are oftenterminated at one or both of their ends near (i.e., within about 5, 10or 20 aa of) the boundaries of functional or structural domains.Fragments terminated at both ends by structural or functional boundariesconsist essentially of a particular segment (or domain) of ACT-4-L aminoacids responsible for a functional or structural property. Structuraland functional domains are identified by comparison of nucleotide and/oramino acid sequence data such as is shown in FIG. 10 to public orproprietary sequence databases. Preferably, computerized comparisonmethods are used to identify sequence motifs or predicted proteinconformation domains that occur in other proteins of known structureand/or function. Binding domains can be identified by epitope mapping.See Section VI, infra. Structural domains include an intracellulardomain, transmembrane domain, and extracellular domain. Functionaldomains include an extracellular binding domain through which ACT-4-Lligand polypeptides interact with external soluble molecules orcell-bound receptors and an intracellular signal-transducing domain.

[0068] Expression of ACT-4-L-h-1 and related ligands is dependent oncell type and activation status. See Example 8. Most notably,ACT-4-L-h-1 is easily detected on some PMA/ionomycin-activated B celllines. ACT-4-L-h-1 is substantially absent on fresh resting B cells.ACT-4-L-h-1 is also expressed on HLTV-1-infected T-cells and may beinfect on other T-cell types in some circumstances. See Example 8.

[0069] The affinity of an ACT-4-L ligand (expressed on activated Bcells) for an ACT-4 receptor (expressed predominantly on activated CD4⁺cells) suggests that the interaction between ligand and receptor mayhave a role in activation/differentiation of CD4⁺ T-cells and/or Bcells. Both CD4⁺ T-cell and B-cell activation are known to be multistepprocesses requiring antigen-specific and nonspecific stimuli. Theinteraction between ACT-4 and ACT-4-L would likely constitute anonantigen-specific stimulus effective on either or both of therespective cells bearing these antigens. The stimulus might be directwhen as, for example, the ligand-receptor binding triggers an enzymicactivity in the intracellular domain in ligand and/or receptor, whichactivity in turn initiates a cascade of metabolic events in one or bothof the respective cells. Alternatively, the stimulus might be indirect;for example, the interaction between ACT-4 and ACT-4-L might increasethe avidity of cellular interactions between other ligand-receptor pairsor control leukocyte localization and migration. Interaction of ACT-4and ACT-4-L may act in conjunction with binding of other T_(H)-B cellreceptor/ligand pairs such as CD2/LFA-3 (Moingeon et al., Nature 339:314(1988)), CD4/MHC class II (Doyle & Stominger, Nature 330:256-259(1987)), LFA-1/ICAM-I/ICAM-2 (Makgoba et al., Nature 331:86-88 (1988)and Staunton et al., Nature 339:61-64 (1989)) and B7/CD28 (Linsley etal., J. Exp. Med. 173:721-730 (1991)). ACT-4/ACT-4-L interactions mayalso be enhanced or diminished by binding of soluble molecules to CD4⁺T-cells and/or B cells. Likely soluble molecules are cytokinesincluding, e.g., interleukins IL-1 through IL-13, tumor necrosis factorsα & β, interferons α, β and γ, tumor growth factor Beta (TGF-β), colonystimulating factor (CSF) and granulocyte monocyte colony stimulatingfactor (GM-CSF).

[0070] Expression of ACT-4-L ligand on certain subtypes of T-cells insome circumstances may confer additional or alternate roles forACT-4/ACT-4-L interactions. For instance, efficiency of infection ofT-cells by human immunodeficiency virus (HIV) or other viruses andpathogens may be affected by ACT-4 or ACT-4-L or by interactions betweenthe two. In addition it is possible that ACT-4 and ACT-4-L could beexpressed by the same cells (e.g., activated CD4⁺ T-cells). In thesecircumstances, interactions between receptor and ligand may affect thegrowth and activation state of their respective cells.

[0071] Fragments or analogs comprising substantially one or morefunctional domain (e.g., an extracellular domain) of ACT-4-L ligandpolypeptides can be fused or otherwise linked to heterologouspolypeptide sequences, such that the resultant fusion protein exhibitsthe functional property(ies) conferred by the ACT-4-L ligand polypeptideand/or the fusion partner. Suitable fusion partners are as discussed inSection I, supra.

[0072] The ACT-4-L ligand polypeptides can be used to affinity purifyrespective ACT-4 receptors. ACT-4-L ligand polypeptides are also usefulas agonists or antagonists of a second ACT-4-L ligand or an ACT-4receptor, and can be used in the therapeutic methods discussed inSection VII, infra. ACT-4 ligand polypeptides are also useful inscreening assays for identifying agonists and antagonists of ACT-4and/or ACT-4-L.

[0073] III. Methods of Producing Polypeptides

[0074] A. Recombinant Technologies

[0075] The nucleotide and amino acid sequences of ACT-4-h-1 shown inFIG. 5, and corresponding sequences for other ACT-4 receptor variantsallow production of polypeptides of full-length ACT-4 receptorpolypeptides sequences and fragments thereof. Similarly, the amino acidsequence of ACT-4-L-h-1 and corresponding sequences for other ACT-4-Lligand polypeptide variants allow production of full-length and fragmentligand polypeptides. Such polypeptides may be produced in prokaryotic oreukaryotic host cells by expression of polynucleotides encoding ACT-4 orACT-4-L, or fragments and analogs of either of these. The cloned DNAsequences are expressed in hosts after the sequences have been operablylinked to (i.e., positioned to ensure the functioning of) an expressioncontrol sequence in an expression vector. Expression vectors aretypically replicable in the host organisms either as episomes or as anintegral part of the host chromosomal DNA. Commonly, expression vectorswill contain selection markers, e.g., tetracycline resistance orhygromycin resistance, to permit detection and/or selection of thosecells transformed with the desired DNA sequences (see, e.g., U.S. Pat.No. 4,704,362).

[0076]E. coli is one prokaryotic host useful for cloning the DNAsequences of the present invention. Other microbial hosts suitable foruse include bacilli, such as Bacillus subtilis, and otherEnterobacteriaceae, such as Salmonella, Serratia, and variousPseudomonas species. In these prokaryotic hosts, one can also makeexpression vectors, which will typically contain expression controlsequences compatible with the host cell (e.g., an origin ofreplication). In addition, any number of a variety of well-knownpromoters will be present, such as the lactose promoter system, atryptophan (trp) promoter system, a beta-lactamase promoter system, or apromoter system from phage lambda. The promoters will typically controlexpression, optionally with an operator sequence, and have ribosomebinding site sequences and the like, for initiating and completingtranscription and translation.

[0077] Other microbes, such as yeast, may also be used for expression.Saccharomyces is a preferred host, with suitable vectors havingexpression control sequences, such as promoters, including3-phosphoglycerate kinase or other glycolytic enzymes, and an origin ofreplication, termination sequences and the like as desired. Insect cells(e.g., SF9) with appropriate vectors, usually derived from baculovirus,are also suitable for expressing ACT-4 receptor or ligand polypeptides.See Luckow, et al. Bio/Technology 6:47-55 (1988) (incorporated byreference for all purposes).

[0078] Higher eukaryotic mammalian tissue cell culture may also be usedto express and produce the polypeptides of the present invention (seeWinnacker, From Genes to Clones (VCH Publishers, NY, N.Y., 1987))(incorporated by reference for all purposes). Eukaryotic cells areactually preferred, because a number of suitable host cell lines capableof secreting and authentically modifying human proteins have beendeveloped in the art, and include the CHO cell lines, various COS celllines, HeLa cells, myeloma cell lines, Jurkat cells, etc. Expressionvectors for these cells can include expression control sequences, suchas an origin of replication, a promoter (e.g., a HSV tk promoter or pgk(phosphoglycerate kinase) promoter), an enhancer (Queen et al., Immunol.Rev. 89:49 (1986)), and necessary processing information sites, such asribosome binding sites, RNA splice sites, polyadenylation sites (e.g.,an SV40 large T Ag poly A addition site), and transcriptional terminatorsequences. Preferred expression control sequences are promoters derivedfrom immunoglobulin genes, SV40, adenovirus, bovine papillomavirus, andthe like. The vectors containing the DNA segments of interest (e.g.,polypeptides encoding an ACT-4 receptor) can be transferred into thehost cell by well-known methods, which vary depending on the type ofcellular host. For example, CaCl₂ transfection is commonly utilized forprokaryotic cells, whereas CaPO₄ treatment or electroporation may beused for other cellular hosts. Vectors may exist as episome orintegrated into the host chromosome.

[0079] B. Naturally Occurring ACT-4 or ACT-4-L Polypeptides

[0080] Natural ACT-4 receptor polypeptides are isolated by conventionaltechniques such as affinity chromatography. For example, polyclonal ormonoclonal antibodies are raised against previously-purified ACT-4-h-1and attached to a suitable affinity column by well known techniques.See, e.g., Hudson & Hay, Practical Immunology (Blackwell ScientificPublications, Oxford, UK, 1980), Chapter 8 (incorporated by referencefor all purposes). For example, anti-ACT-4-h-1 can be immobilized to aprotein-A sepharose column via crosslinking of the F_(c) domain with ahomobifunctional crosslinking agent, such as dimethyl pimelimidate. Cellextracts are then passed through the column, and ACT-4 receptor proteinspecifically bound by the column, eluted with, for example, 0.5 Mpyrogenic acid, pH 2.5. Usually, an intact form of ACT-4 receptor isobtained by such isolation techniques. Peptide fragments are generatedfrom intact ACT-4 receptors by chemical (e.g., cyanogen bromide) orenzymatic cleavage (e.g., V8 protease or trypsin) of the intactmolecule.

[0081] Naturally occurring ACT-4-L ligand polypeptides can be purifiedusing an analogous approach except that the affinity reagent is anantibody specific for ACT-4-L-h-1.

[0082] C. Other Methods

[0083] Alternatively, ACT-4 or ACT-4-L polypeptides can be synthesizedby chemical methods or produced by in vitro translation systems using apolynucleotide template to direct translation. Methods for chemicalsynthesis of polypeptides and in vitro translation are well known in theart, and are described further by Berger & Kimmel, Methods inEnzymology, Volume 152, Guide to Molecular Cloning Techniques AcademicPress, Inc., San Diego, Calif., 1987).

[0084] IV. Nucleic Acids

[0085] A. Cloning ACT-4 or ACT-4-L Nucleic Acids

[0086] Example 5 presents nucleic acid sequence data for a cDNA clone ofan ACT-4 receptor designated ACT-4-h-1. The sequence includes both atranslated region and 3′ and 5′ flanking regions. This sequence data canbe used to design probes with which to isolate other ACT-4 receptorgenes. These genes include the human genomic gene encoding ACT-4-h-1,and cDNAs and genomic clones from higher mammalian species, and allelicand nonallelic variants, and natural and induced mutants of all of thesegenes. Specifically, all nucleic acid fragments encoding all ACT-4receptor polypeptides disclosed in this application are provided.Genomic libraries of many species are commercially available (e.g.,Clontech, Palo Alto, Calif.), or can be isolated de novo by conventionalprocedures. cDNA libraries are best prepared from activated CD4⁺ cells,which express ACT-4-h-1 in large amounts.

[0087] The probes used for isolating clones typically comprise asequence of about at least 24 contiguous nucleotides (or theircomplement) of the cDNA sequence shown in FIG. 5. For example, afull-length polynucleotide corresponding to the sequence shown in FIG. 5can be labeled and used as a hybridization probe to isolate genomicclones from a human genomic clone library in e.g., λEMBL4 or λGEM11(Promega Corporation, Madison, Wis.); typical hybridization conditionsfor screening plaque lifts (Benton & Davis, Science 196:180 (1978)) canbe: 50% formamide, 5× SSC or SSPE, 1-5× Denhardt's solution, 0.1-1% SDS,100-200 μg sheared heterologous DNA or tRNA, 0-10% dextran sulfate,1×10⁵ to 1×10⁷ cpm/ml of denatured probe with a specific activity ofabout 1×10⁸ cpm/μg, and incubation at 42° C. for about 6-36 hours.Prehybridization conditions are essentially identical except that probeis not included and incubation time is typically reduced. Washingconditions are typically 1-3× SSC, 0.1-1% SDS, 50-70° C. with change ofwash solution at about 5-30 minutes. Hybridization and washingconditions are typically less stringent for isolation of higher cognateor nonallelic variants than for e.g., the human genomic clone ofACT-4-h-1.

[0088] Alternatively, probes can be used to clone ACT-4 receptor genesby methods employing the polymerase chain reaction (PCR). Methods forPCR amplification are described in e.g., PCR Technology: Principles andApplications for DNA Amplification (ed. H. A. Erlich, Freeman Press, NY,N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (eds.Innis, et al., Academic Press, San Diego, Calif., 1990); Mattila et al.,Nucleic Acids Res. 19:4967 (1991); Eckert, K. A. and Kunkel, T. A., PCRMethods and Applications 1:17 (1991); PCR (eds. McPherson et al., IRLPress, Oxford); and U.S. Pat. No. 4,683,202 (each of which isincorporated by reference for all purposes).

[0089] Alternatively, synthetic polynucleotide sequences correspondingto all or part of the sequences shown in FIG. 5 may be constructed bychemical synthesis of oligonucleotides.

[0090] Nucleotide substitutions, deletions, and additions can beincorporated into the polynucleotides of the invention. Nucleotidesequence variation may result from degeneracy of the genetic code, fromsequence polymorphisms of various ACT-4 receptor alleles, minorsequencing errors, or may be introduced by random mutagenesis of theencoding nucleic acids using irradiation or exposure to EMS, or bychanges engineered by site-specific mutagenesis or other techniques ofmodern molecular biology. See Sambrook et al., Molecular Cloning: ALaboratory Manual (C.S.H.P. Press, NY 2d ed., 1989) (incorporated byreference for all purposes). For nucleotide sequence that are capable ofbeing transcribed and translated to produce a functional polypeptide,degeneracy of the genetic code results in a number of nucleotidesequences that encode the same polypeptide. The invention includes allsuch sequences. Generally, nucleotide substitutions, deletions, andadditions should not substantially disrupt the ability of an ACT-4receptor polynucleotide to hybridize to the sequence of ACT-4-h-1 shownin FIG. 5 under stringent conditions. Typically, ACT-4 receptorpolynucleotides comprise at least 25 consecutive nucleotides which aresubstantially identical to a naturally-occurring ACT-4 receptor sequence(e.g., FIG. 5), more usually ACT-4 receptor polynucleotides comprise atleast 50 to 100 consecutive nucleotides, which are substantiallyidentical to a naturally-occurring ACT-4 receptor sequence.

[0091] ACT-4 receptor polynucleotides can be short oligonucleotides(e.g., about 10, 15, 25, 50 or 100 contiguous bases from the ACT-h-1sequence shown in FIG. 5), such as for use as hybridization probes andPCR (or LCR) primers. ACT-4 receptor polynucleotide sequences can alsocomprise part of a larger polynucleotide that includes sequences thatfacilitate transcription (expression sequences) and translation of thecoding sequences, such that the encoded polypeptide product is produced.Construction of such polynucleotides is well known in the art and isdescribed further in Sambrook et al., supra (C.S.H.P. Press, NY 2d ed.1989). The ACT-4 receptor polynucleotide can be fused in frame withanother polynucleotide sequence encoding a different protein (e.g.,glutathione S-transferase, β-galactosidase or an immunoglobulin F_(C)domain) for encoding expression of a fusion protein (see, e.g., Byrn etal., Nature, 344:667-670 (1990)) (incorporated by reference for allpurposes).

[0092] Nucleic acids encoding ACT-4-L genes can be produced by ananalogous approach, using the ACT-4-L-h-1 cDNA sequence shown in FIG. 10as a starting material for probe design. ACT-4-L genes include the humangenomic gene encoding ACT-4-L-h-1, allelic and nonallelic variants,higher cognate variants, and natural and induced mutants of all of thesegenes. Specifically, all nucleic acid fragments (genomic, cDNA orsynthetic) encoding all ACT-4-L polypeptides disclosed in thisapplication are provided. In nucleic acid fragments having mutations ofnaturally occurring sequences, generally the mutation will notsubstantially disrupt the ability of an ACT-4-L polynucleotide tohybridize to the nucleotide sequence of ACT-4-L-h-1 under stringentconditions. ACT-4-L ligand polynucleotides can be short oligonucleotides(e.g., about 10, 15, 25, 50 or 100 contiguous bases from the ACT-4-L-h-1sequence shown in FIG. 10), such as for use as hybridization probes andPCR (or LCR) primers. ACT-4-L ligand polynucleotide sequences can alsocomprise part of a larger polynucleotide as discussed in connection withACT-4 polynucleotide sequences.

[0093] V. Antibodies and Hybridomas

[0094] In another embodiment of the invention, antibodies against ACT-4and ACT-4-L polypeptides are provided.

[0095] A. General Characteristics of Antibodies

[0096] Antibodies or immunoglobulins are typically composed of fourcovalently bound peptide chains. For example, an IgG antibody has twolight chains and two heavy chains. Each light chain is covalently boundto a heavy chain. In turn each heavy chain is covalently linked to theother to form a “Y” configuration, also known as an immunoglobulinconformation. Fragments of these molecules, or even heavy or lightchains alone, may bind antigen. Antibodies, fragments of antibodies, andindividual chains are also referred to herein as immunoglobulins.

[0097] A normal antibody heavy or light chain has an N-terminal (NH₂)variable (V) region, and a C-terminal (—COOH) constant (C) region. Theheavy chain variable region is referred to as V_(H) (including, forexample, V_(γ)), and the light chain variable region is referred to asV_(L) (including V_(κ) or V_(λ)). The variable region is the part of themolecule that binds to the antibody's cognate antigen, while the Fcregion (the second and third domains of the C region) determines theantibody's effector function (e.g., complement fixation, opsonization).Full-length immunoglobulin or antibody “light chains” (generally about25 kDa, about 214 amino acids) are encoded by a variable region gene atthe N-terminus (generally about 110 amino acids) and a κ (kappa) or λ(lambda) constant region gene at the COOH-terminus. Full-lengthimmunoglobulin or antibody “heavy chains” (generally about 50 Kd, about446 amino acids), are similarly encoded by a variable region gene(generally encoding about 116 amino acids) and one of the constantregion genes, e.g., gamma (encoding about 330 amino acids). Typically,the “V_(L)” will include the portion of the light chain encoded by theV_(L) and/or J_(L) (J or joining region) gene segments, and the “V_(H)”will include the portion of the heavy chain encoded by the V_(H), and/orD_(H) (D or diversity region) and J_(H) gene segments. See, generally,Roitt et al., Immunology (2d ed. 1989), Chapter 6 and Paul, FundamentalImmunology (Raven Press, 2d ed., 1989) (each of which is incorporated byreference for all purposes).

[0098] An immunoglobulin light or heavy chain variable region consistsof a “framework” region interrupted by three hypervariable regions, alsocalled complementarity-determining regions or CDRs. The extent of theframework region and CDRs have been defined (see Kabat et al. (1987),“Sequences of Proteins of Immunological Interest,” U.S. Department ofHealth and Human Services; Chothia et al., J. Mol. Biol. 196:901-917(1987) (each of which is incorporated by reference for all purposes).The sequences of the framework regions of different light or heavychains are relatively conserved within a species. The framework regionof an antibody, that is the combined framework regions of theconstituent light and heavy chains, serves to position and align theCDRs in three dimensional space. The CDRs are primarily responsible forbinding to an epitope of an antigen. The CDRs are typically referred toas CDR1, CDR2, and CDR3, numbered sequentially starting from theN-terminus.

[0099] The constant region of the heavy chain molecule, also known asC_(H), determines the isotype of the antibody. Antibodies are referredto as IgM, IgD, IgG, IgA, and IgE depending on the heavy chain isotype.The isotypes are encoded in the mu (μ), delta (Δ), gamma (γ), alpha (α),and epsilon (ε) segments of the heavy chain constant region,respectively. In addition, there are a number of γ subtypes. There aretwo types of light chains, κ and λ. The determinants of these subtypestypically reside in the constant region of the light chain, alsoreferred to as the C_(L) in general, and C_(κ) or C_(λ) in particular.

[0100] The heavy chain isotypes determine different effector functionsof the antibody, such as opsonization or complement fixation. Inaddition, the heavy chain isotype determines the secreted form of theantibody. Secreted IgG, IgD, and IgE isotypes are typically found insingle unit or monomeric form. Secreted IgM isotype is found inpentameric form; secreted IgA can be found in both monomeric and dimericform.

[0101] B. Production of Antibodies

[0102] Antibodies which bind either an ACT-4 receptor, an ACT-4-Lligand, or binding fragments of either, can be produced by a variety ofmeans. The production of nonhuman monoclonal antibodies, e.g., murine,rat and so forth, is well known and may be accomplished by, for example,immunizing the animal with a preparation containing an ACT-4 receptor orits ligands, or an immunogenic fragment of either of these.Particularly, useful as immunogens are cells stably transfected with arecombinant ACT-4 or ACT-4-L gene and expressing an ACT-4 receptor orligand thereto on their cell surface. Antibody-producing cells obtainedfrom the immunized animals are immortalized and screened for theproduction of an antibody which binds to ACT-4 receptors or theirligands. See Harlow & Lane, Antibodies, A Laboratory Manual (C.S.H.P.NY, 1988) (incorporated by reference for all purposes). A number ofmurine antibodies to a protein having a substantially similar oridentical primary amino acid sequence to ACT-4-L-h-1 have been discussedby Tozawa et al., Int. J. Cancer 41:231-238 (1988); Tanaka et al., Int.J. Cancer 36:549-555 (1985) (incorporated by reference in their entiretyfor all purposes).

[0103] Several techniques for generation of human monoclonal antibodieshave also been described but are generally more onerous than murinetechniques and not applicable to all antigens. See, e.g., Larrick etal., U.S. Pat. No. 5,001,065, for review (incorporated by reference forall purposes). One technique that has successfully been used to generatehuman monoclonal antibodies against a variety of antigens is the triomamethodology of Ostberg et al. (1983), Hybridoma 2:361-367, Ostberg, U.S.Pat. No. 4,634,664, and Engleman et al., U.S. Pat. No. 4,634,666(incorporated by reference for all purposes). The antibody-producingcell lines obtained by this method are called triomas, because they aredescended from three cells—two human and one mouse. Triomas have beenfound to produce antibody more stably than ordinary hybridomas made fromhuman cells.

[0104] An alternative approach is the generation of humanizedimmunoglobulins by linking the CDR regions of nonhuman antibodies tohuman constant regions by recombinant DNA techniques. See Queen et al.,Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989) and WO 90/07861(incorporated by reference for all purposes). The humanizedimmunoglobulins have variable region framework residues substantiallyfrom a human immunoglobulin (termed an acceptor immunoglobulin) andcomplementarity determining regions substantially from a mouseimmunoglobulin, e.g., the L106 antibody (see Example 1) (referred to asthe donor immunoglobulin). The constant region(s), if present, are alsosubstantially from a human immunoglobulin. The human variable domainsare usually chosen from human antibodies whose framework sequencesexhibit a high degree of sequence identity with the murine variableregion domains from which the CDRs were derived. The heavy and lightchain variable region framework residues can be derived from the same ordifferent human antibody sequences. The human antibody sequences can bethe sequences of naturally occurring human antibodies or can beconsensus sequences of several human antibodies. See Carter et al., WO92/22653. Certain amino acids from the human variable region frameworkresidues are selected for substitution based on their possible influenceon CDR conformation and/or binding to antigen. Investigation of suchpossible influences is by modeling, examination of the characteristicsof the amino acids at particular locations, or empirical observation ofthe effects of substitution or mutagenesis of particular amino acids.

[0105] For example, when an amino acid differs between a murine L106variable region framework residue and a selected human variable regionframework residue, the human framework amino acid should usually besubstituted by the equivalent framework amino acid from the mouseantibody when it is reasonably expected that the amino acid:

[0106] (1) noncovalently binds antigen directly,

[0107] (2) is adjacent to a CDR region,

[0108] (3) otherwise interacts with a CDR region (e.g., is within about3 Å of a CDR region), or

[0109] (4) participates in the V_(L)-V_(H) interface.

[0110] Other candidates for substitution are acceptor human frameworkamino acids that are unusual for a human immunoglobulin at thatposition. These amino acids can be substituted with amino acids from theequivalent position of the L106 antibody or from the equivalentpositions of more typical human immunoglobulins.

[0111] A further approach for isolating DNA sequences which encode ahuman monoclonal antibody or a binding fragment thereof is by screeninga DNA library from human B cells according to the general protocoloutlined by Huse et al., Science 246:1275-1281 (1989) and then cloningand amplifying the sequences which encode the antibody (or bindingfragment) of the desired specificity. The protocol described by Huse isrendered more efficient in combination with phage display technology.See, e.g., Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047.Phage display technology can also be used to mutagenize CDR regions ofantibodies previously shown to have affinity for ACT-4 receptors ortheir ligands. Antibodies having improved binding affinity are selected.

[0112] Anti-ACT-4 receptor antibodies that specifically bind to the sameepitope as the L106 antibody are usually identified by a competitivebinding assay. The assay has three components, an ACT-4 polypeptide(e.g., ACT-4-h-1), L106 antibody, which is usually labelled, and theantibody under test. Often the ACT-4 receptor polypeptide is immobilizedto a solid support. The test antibody binds to the same epitope as theL106 antibody if it reduces the amount of L106 antibody thatspecifically binds to the ACT-4 receptor polypeptide. The extent ofscreening necessary to obtain such antibodies can be reduced bygenerating antibodies with a protocol in which the specific epitopebound by L106 is used as an immunogen. Antibodies binding to the sameepitope as L106 may exhibit a substantially, but not completely,identical amino acid sequence to the L106 antibody, or may have anunrelated primary structure to the L106 antibody.

[0113] Anti-ACT-4 receptor antibodies having a different bindingspecificity than L106 (i.e., which bind to a different epitope) areidentified by a complementary approach. Test antibodies are screened forfailure to compete with the L106 antibody for binding to an ACT-4receptor polypeptide. The extent of screening can be reduced bygenerating antibodies with a protocol in which a fragment lacking aspecific epitope bound by L106 is used as an immunogen.

[0114] Antibodies having the same or different binding specificity to aselected antibody to an ACT-4-L ligand polypeptide can be identified byanalogous procedures.

[0115] Antibodies having the capacity to stimulate or inhibit activationof CD4⁺ or B cells can be identified by the screening proceduresdiscussed in Section VI, infra. Some antibodies may selectively inhibitactivation in response to some stimuli (e.g., alloantigenic but notmitogenic, or vice versa), and not to others. Some antibodies'inhibitory capacity may depend on the time after activation at which theantibody is added. Some antibodies may have the capacity to activateCD4⁺ or B cells independently of other stimuli, whereas other antibodiesmay only have the capacity to augment the efficacy of another stimulussuch as that provided by PHA or PMA/ionomycin.

[0116] Antibodies isolated by the above procedures can be used togenerate anti-idiotypic antibodies by, for example, immunization of ananimal with the primary antibody. For anti-ACT-4 receptor antibodies,anti-idiotype antibodies whose binding to the primary antibody isinhibited by ACT-4 receptors or fragments thereof are selected. Becauseboth the anti-idiotypic antibody and the ACT-4 receptors or fragmentsthereof bind the primary immunoglobulin, the anti-idiotypicimmunoglobulin may represent the “internal image” of an epitope and thusmay substitute for the ACT-4-L ligand polypeptide. Anti-idiotypicantibodies to an ACT-4-L ligand polypeptide that can substitute for anACT-4 receptor can be produced by an analogous approach.

[0117] C. Epitope Mapping

[0118] The epitope bound by the L106 antibody or any other selectedantibody to an ACT-4 receptor is determined by providing a family offragments containing different amino acid segments from an ACT-4receptor polypeptide, such as ACT-4-h-1. Each fragment typicallycomprises at least 4, 6, 8, 10, 20, 50 or 100 contiguous amino acids.Collectively, the family of polypeptide covers much or all of the aminoacid sequence of a full-length ACT-4 receptor polypeptide. Members ofthe family are tested individually for binding to e.g., the L106antibody. The smallest fragment that can specifically bind to theantibody under test delineates the amino acid sequence of the epitoperecognized by the antibody. An analogous approach is used to mapepitopes bound by antibodies to the ACT-4 ligand polypeptides.

[0119] D. Fragments of Antibodies, and Immunotoxins

[0120] In another embodiment of the invention, fragments of antibodiesagainst ACT-4 receptors or their ligands are provided. Typically, thesefragments exhibit specific binding to the ACT-4 receptor or ligand withan affinity of at least 10⁷ M, and more typically 10⁸ or 10⁹ M. Antibodyfragments include separate heavy chains, light chains Fab, Fab′ F(ab′)₂,Fabc, and Fv. Fragments are produced by recombinant DNA techniques, orby enzymic or chemical separation of intact immunoglobulins.

[0121] In another embodiment, immunotoxins are provided. An immunotoxinis a chimeric compound consisting of a toxin linked to an antibodyhaving a desired specificity. The antibody serves as a targeting agentfor the toxin. See generally Pastan et al., Cell 47:641-648 (1986). Atoxin moiety is couple to an intact antibody or a fragment thereof bychemical or recombinant DNA techniques. Preferably, the toxin is linkedto an immunoglobulin chain in the form of a contiguous protein. See,e.g., Chovnick et al., Cancer Res. 51:465; Chaudhary et al., Nature339:394 (1989) (incorporated by reference for all purposes). Examples ofsuitable toxin components are listed in Section I, supra, and arereviewed in, e.g., The Specificity and Action of Animal, Bacterial andPlant Toxins (ed. P. Cuatrecasas, Chapman Hall, London, 1976)(incorporated by reference for all purposes).

[0122] E. Hybridomas and Other Cell Lines

[0123] All hybridomas, triomas and other cell lines producing theantibodies and their fragments discussed, supra, are expressly includedin the invention. These include the hybridoma line HBL106, depositedunder the Budapest Treaty at the American Type Culture Collection,Rockville, Md. 20852 as ATCC HB11483, which produces the L106 mouseantibody.

[0124] F. Uses of Antibodies

[0125] Antibodies to ACT-4 and ACT-4-L polypeptides and their bindingfragments are useful for screening cDNA expression libraries, preferablycontaining human or primate cDNA derived from various tissues and foridentifying clones containing cDNA inserts, which encodestructurally-related, immunocrossreactive proteins. See Aruffo & Seed,Proc. Natl. Acad. Sci. USA 84:8573-8577 (1987) (incorporated byreference for all purposes). Antibodies are also useful to identifyand/or purify immunocrossreactive proteins that are structurally orevolutionarily related to the native ACT-4 receptor or ACT-4-L ligandpolypeptides or to fragments thereof used to generate the antibody.Diagnostic and therapeutic uses of antibodies, binding fragmentsthereof, immunotoxins and idiotypic antibodies are described in SectionVII, infra.

[0126] VI. Screening for Agonists and Antagonists

[0127] ACT-4 and ACT-4-L polypeptides, fragments, analogs thereof,antibodies and anti-idiotypic antibodies thereto, as well as otherchemical or biological agents are screened for their ability to block orenhance binding of an ACT-4 to its ligand. In addition, they are testedfor their ability to stimulate or inhibit metabolic processes, such asDNA synthesis or protein phosphorylation in cells bearing either anACT-4 receptor or an ACT-4-L ligand polypeptide anchored to theirsurfaces.

[0128] In some methods, the compound under test is screened for itsability to block or enhance binding of a purified binding fragment of anACT-4 receptor (or fusion protein thereof) to a purified bindingfragment of an ACT-4-L ligand polypeptide (or fusion protein thereof).In such experiments, either the receptor or ligand fragment is usuallyimmobilized to a solid support. The test compound then competes with anACT-4 or ACT-4-L fragment (whichever is not attached to the support) forbinding to the support. Usually, either the test compound or thecompeting ligand or receptor is labelled.

[0129] In other methods, either or both of the ACT-4 receptor andACT-4-L ligand polypeptide, or binding fragments of these molecules, areexpressed on a cell surface. For example, an ACT-4-L ligand polypeptidemay be expressed on activated B-cells and/or an ACT-4 receptorpolypeptide expressed on activated CD4⁺ T-cells. Alternatively, eitherthe ligand or receptor can be expressed from recombinant DNA in e.g.,COS-7 cells (see Example 6). In these methods, the existence of agonismor antagonism is determined from the degree of binding between an ACT-4receptor and its ligand that occurs in the presence of the testcompound.

[0130] Alternatively, activity of the test compound is assayed bymeasurement of ³H-thymidine incorporation into DNA or ³²P-incorporationinto proteins in cells bearing an ACT-4 receptor and/or cells bearing anACT-4-L ligand polypeptide.

[0131] Compounds that block ACT-4 or ACT-4-L polypeptide-induced DNAsynthesis or protein phosphorylation are antagonists. Compounds thatactivate DNA synthesis or phosphorylation via interaction with an ACT-4receptor or its ligands are agonists. Agonistic or antagonistic activitycan also be determined from other functional or physical endpoints ofleukocyte activation, or from clinically desirable or undesirableoutcomes, such as cytolytic activity, or extravasation of leukocytesinto tissues from blood vessels.

[0132] The ability of agents to agonize or antagonize T-cellproliferation in vitro can be correlated with the ability to affect theimmune response in vivo. In vivo activity is typically assayed usingsuitable animal models such as mice or rats. To assay the effect ofagents on allograft rejection, for example, potential therapeutic agentscan be administered to the animals at various times before introductionof the allogeneic tissue; and the animals can be monitored for graftrejection. Suitable methods for performing the transplant and monitoringfor graft rejection have been described (see, e.g., Hislop et al., J.Thorac. Cardiovasc. 100:360-370 (1990)) (incorporated by reference forall purposes).

[0133] VII. Therapeutic and Diagnostic Methods and Compositions

[0134] A. Diagnostic Methods

[0135] Diseases and conditions of the immune system associated with analtered abundance, or functional mutation, of an ACT-4 receptor or itsmRNA, or an ACT-4-L ligand or its mRNA may be diagnosed using the probesand/or antibodies of the present invention. Detection of an ACT-4receptor or mRNA allows activated CD4⁺ T-cells to be distinguished fromother leukocyte subtypes. For example, ACT-4 receptor can be detectedusing an antibody or an ACT-4-L polypeptide that specifically binds tothe ACT-4 receptor. The presence of activated CD4⁺ T-cells is indicativeof a MHC class II induced immune response against, e.g., invadingbacteria. Comparison of the numbers of activated CD4⁺ cells and CD8⁺cells may allow differential diagnosis between bacterial and viralinfections, which predominantly induce these respective activated celltypes. The presence of activated CD4⁺ cells is also indicative ofundesirable diseases and conditions of the immune system, such asallograft rejection, graft versus host disease, autoimmune diseases,allergies and inflammation. The efficacy of therapeutic agents intreating such diseases and conditions can be monitored.

[0136] Detection of ACT-4-L ligand or its mRNA may indicate the presenceof activated B-cells and/or signal that the appearance of activated CD4⁺T-cells is imminent. ACT-4-L ligand can, for example, be detected usingan antibody or an ACT-4 receptor polypeptide that specifically binds tothe ACT-4-L ligand. Successive detection of ACT-4-L ligand followed byACT-4 receptor (or vice versa) can allow monitoring of a progressionthrough different temporal stages of activation in an immune response.

[0137] Diagnosis can be accomplished by removing a cellular sample(e.g., blood sample, lymph node biopsy or tissue) from a patient. Thesample is then subjected to analysis for determining: (1) the amount ofexpressed ACT-4 receptor or ACT-4-L ligand in individual cells of thesample (e.g., by immunohistochemical staining of fixed cells with anantibody or FACS™ analysis), (2) the amount of ACT-4 receptor or ACT-4-Lligand mRNA in individual cells (by in situ hybridization with alabelled complementary polynucleotide probe), (3) the amount of ACT-4receptor or ACT-4-L ligand mRNA in the cellular sample by RNA extractionfollowed by hybridization to a labeled complementary polynucleotideprobe (e.g., by Northern blotting, dot blotting, solution hybridizationor quantitative PCR), or (4) the amount of ACT-4 receptor or ACT-4-Lligand in the cellular sample (e.g., by cell disruption followed byimmunoassay or Western blotting of the resultant cell extract).

[0138] Diagnosis can also be achieved by in vivo administration of adiagnostic reagent (e.g., a labelled anti-ACT-4 or ACT-4-L antibody formonitoring of activated CD4⁺ T-cells or B-cells, respectively) anddetection by in vivo imaging. The concentration of diagnostic agentadministered should be sufficient that the binding to those cells havingthe target antigen is detectable compared to the background signal.Further, it is desirable that the diagnostic reagent can be rapidlycleared from the circulatory system in order to give the besttarget-to-background signal ratio. The diagnostic reagent can belabelled with a radioisotope for camera imaging, or a paramagneticisotope for magnetic resonance or electron spin resonance imaging.

[0139] A change (typically an increase) in the level of protein or mRNAof an ACT-4 receptor or ACT-4-L ligand in a cellular sample from anindividual, which is outside the range of clinically established normallevels, may indicate the presence of an undesirable immune reaction inthe individual from whom the sample was obtained, and/or indicate apredisposition of the individual for developing (or progressing through)such a reaction. Protein or mRNA levels may be employed as adifferentiation marker to identify and type cells of certain lineages(e.g., activated CD4⁺ cells for the ACT-4 receptor) and developmentalorigins. Such cell-type specific detection may be used forhistopathological diagnosis of undesired immune responses.

[0140] B. Diagnostic Kits

[0141] In another aspect of the invention, diagnostic kits are providedfor the diagnostic methods described supra. The kits comprisecontainer(s) enclosing the diagnostic reagents, such as labelledantibodies against ACT-4 receptors and ACT-4-L ligands, and reagentsand/or apparatus for detecting the label. Other components routinelyfound in such kits may also be included together with instructions forperforming the diagnostic test.

[0142] C. Pharmaceutical Compositions

[0143] The pharmaceutical compositions used for prophylactic ortherapeutic treatment comprise an active therapeutic agent, for example,an ACT-4 receptor, an ACT-4-L ligand, fragments thereof, and antibodiesand idiotypic antibodies thereto, and a variety of other components. Thepreferred form depends on the intended mode of administration andtherapeutic application. The compositions may also include, depending onthe formulation desired, pharmaceutically-acceptable, non-toxic carriersor diluents, which are defined as vehicles commonly used to formulatepharmaceutical compositions for animal or human administration. Thediluent is selected so as not to affect the biological activity of thecombination. Examples of such diluents are distilled water,physiological saline, Ringer's solutions, dextrose solution, and Hank'ssolution. In addition, the pharmaceutical composition or formulation mayalso include other carriers, adjuvants, or nontoxic, nontherapeutic,nonimmunogenic stabilizers and the like.

[0144] D. Therapeutic Methods

[0145] The therapeutic methods employ the therapeutic agents discussedabove for treatment of various diseases in humans or animals,particularly vertebrate mammals. The therapeutic agents include ACT-4receptors, binding fragments thereof, ACT-4-L ligands, binding fragmentsthereof, anti-ACT-4 receptor and anti-ACT-4-L ligand antibodies andanti-idiotypic antibodies thereto, binding fragments of theseantibodies, humanized versions of these antibodies, immunotoxins, andother agents discussed, supra. Some therapeutic agents function byblocking or otherwise antagonizing the action of an ACT-4 receptor withits ligand. Preferred therapeutic agents compete with the ligand forbinding to the receptor, or compete with the receptor for binding to theligand. Other therapeutic agents function by killing cells bearing apolypeptide against which the agent is targeted. For example, anti-ACT-4receptor antibodies with effector functions or which are conjugated totoxins, radioisotopes or drugs are capable of selectively killingactivated CD4⁺ T-cells. Similarly, anti-ACT-4-L ligand antibodies arecapable of killing activated B cells under analogous circumstances.Selective elimination of activated cells is particularly advantageousbecause an undesirable immune response can be reduced or eliminated,while preserving a residual immune capacity in the form of inactivatedB-cells, CD4⁺ cells and CD8⁺ cells to combat invading microorganisms towhich a patient may subsequently be exposed. Other therapeutic agentsfunction as agonists of the interaction between an ACT-4 receptor andACT-4-L ligand.

[0146] 1. Dosages and Methods of Administration

[0147] In therapeutic applications, a pharmaceutical composition (e.g.,comprising an antibody to ACT-4-h-1 or ACT-4-L-h-1) is administered, invivo or ex vivo, to a patient already suffering from an undesirableimmune response (e.g., transplant rejection), in an amount sufficient tocure, partially arrest, or detectably slow the progression of thecondition, and its complications. An amount adequate to accomplish thisis defined as a “therapeutically effective dose” or “efficacious dose.”Amounts effective for this use will depend upon the severity of thecondition, the general state of the patient, and the route ofadministration, and combination with other immunosuppressive drugs, ifany, but generally range from about 10 ng to about 1 g of active agentper dose, with single dosage units of from 10 mg to 100 mg per patientbeing commonly used. Pharmaceutical compositions can be administeredsystemically by intravenous infusion, or locally by injection. Thelatter is particularly useful for localized undesired immune responsesuch as host versus graft rejection. For a brief review of methods fordrug delivery, see Langer, Science 249:1527-1533 (1990) (incorporated byreference for all purposes).

[0148] In prophylactic applications, pharmaceutical compositions areadministered to a patients at risk of, but not already suffering anundesired immune reaction (e.g., a patient about to undergo transplantsurgery). The amount of agents to be administered is a “prophylacticallyeffective dose,” the precise amounts of which will depend upon thepatient's state of health and general level of immunity, but generallyrange from 10 ng to 1 g per dose, especially 10 mg to 100 mg perpatient.

[0149] Because the therapeutic agents of the invention are likely to bemore selective and generally less toxic than conventionalimmunomodulating agents, they will be less likely to cause the sideeffects frequently observed with the conventional agents. Moreover,because some of the therapeutic agents are human protein sequences(e.g., binding fragments of an ACT-4 receptor or ACT-4 ligand orhumanized antibodies), they are less likely to cause immunologicalresponses such as those observed with murine anti-CD3 antibodies. Thetherapeutic agents of the present invention can be combined with eachother. For example, a combination of antibodies against an ACT-4-Lligand with antibodies against an ACT-4 receptor is likely to provide aparticularly effective blockade against T-cell activation. The agents ofthe invention can also be combined with traditional therapeutics, andcan be used to lower the dose of such agents to levels below thoseassociated with side effects. For example, other immunosuppressiveagents such as antibodies to the α3 domain, T cell antigens (e.g., OKT4and OKT3, CD28), B-cell antigen (B7 or B7-2), antithymocyte globulin, aswell as chemotherapeutic agents such as cyclosporine, glucocorticoids,azathioprine, prednisone can be used in conjunction with the therapeuticagents of the present invention.

[0150] For destruction of a specific population of target cells, it canbe advantageous to conjugate the therapeutic agents of the presentinvention to another molecule. For example, the agents can be joined toliposomes containing particular immunosuppressive agents, to a specificmonoclonal antibody or to a cytotoxin or other modulator of cellularactivity, whereby binding of the conjugate to a target cell populationwill result in alteration of that population. A number of protein toxinshave been discussed supra. Chemotherapeutic agents include, for example,doxorubicin, daunorubicin, methotrexate, cytotoxin, and anti-sense RNA.Antibiotics can also be used. In addition, radioisotopes such asyttrium-90, phosphorus-32, lead-212, iodine-131, or palladium-109 can beused. The emitted radiation destroys the targeted cells.

[0151] 2. Diseases and Conditions Amenable to Treatment

[0152] The pharmaceutical compositions discussed above are suitable fortreating several diseases and conditions of the immune system.

[0153] a. Transplant Rejection

[0154] Over recent years there has been a considerable improvement inthe efficiency of surgical techniques for transplanting tissues andorgans such as skin, kidney, liver, heart, lung, pancreas and bonemarrow. Perhaps the principal outstanding problem is the lack ofsatisfactory agents for inducing immunotolerance in the recipient to thetransplanted allograft or organ. When allogeneic cells or organs aretransplanted into a host (i.e., the donor and donee are differentindividual from the same species), the host immune system is likely tomount an immune response to foreign antigens in the transplant(host-versus-graft disease) leading to destruction of the transplantedtissue. CD8⁺ cells, CD4⁺ cells and monocytes are all involved in therejection of transplant tissues. The therapeutic agents of the presentinvention are useful to block alloantigen-induced immune responses inthe donee (e.g., blockage or elimination of allogen-activation of CD4⁺T-cells by antibodies to an ACT-4 receptor or ACT-4-L ligand) therebypreventing such cells from participating in the destruction of thetransplanted tissue or organ.

[0155] b. Graft Versus Host Disease

[0156] A related use for the therapeutic agents of the present inventionis in modulating the immune response involved in “graft versus host”disease (GVHD). GVHD is a potentially fatal disease that occurs whenimmunologically competent cells are transferred to an allogeneicrecipient. In this situation, the donor's immunocompetent cells mayattack tissues in the recipient. Tissues of the skin, gut epithelia andliver are frequent targets and may be destroyed during the course ofGVHD. The disease presents an especially severe problem when immunetissue is being transplanted, such as in bone marrow transplantation;but less severe GVHD has also been reported in other cases as well,including heart and liver transplants. The therapeutic agents of thepresent invention are used to block activation of, or eliminate, thedonor leukocytes (particularly activated CD4⁺ T-cells and B-cells),thereby inhibiting their ability to lyse target cells in the host.

[0157] c. Autoimmune Diseases

[0158] A further situation in which immune suppression is desirable isin treatment of autoimmune diseases such as insulin-dependent diabetesmellitus, multiple sclerosis, stiff man syndrome, rheumatoid arthritis,myasthenia gravis and lupus erythematosus. In these disease, the bodydevelops a cellular and/or humoral immune response against one of itsown antigens leading to destruction of that antigen, and potentiallycrippling and/or fatal consequences. Activated CD4⁺ T-cells are believedto play a major role in many autoimmune diseases such as diabetesmellitus. Activated B cells play a major role in other autoimmunediseases such as stiff man syndrome. Autoimmune diseases are treated byadministering one of the therapeutic agents of the invention, whichblock activation of, and/or eliminate CD4⁺ T-cells and/or B-cells.Optionally, the autoantigen, or a fragment thereof, against which theautoimmune disease is targeted can be administered shortly before,concurrently with, or shortly after the immunosuppressive agent. In thismanner, tolerance can be induced to the autoantigen under cover of thesuppressive treatment, thereby obviating the need for continuedimmunosuppression. See, e.g., Cobbold et al., WO 90/15152 (1990).

[0159] d. Inflammation

[0160] Inflammation represents the consequence of capillary dilationwith accumulation of fluid and migration of phagocytic leukocytes, suchas granulocytes and monocytes. Inflammation is important in defending ahost against a variety of infections but can also have undesirableconsequences in inflammatory disorders, such as anaphylactic shock,arthritis, gout and ischemia-reperfusion. Activated T-cells have animportant modulatory role in inflammation, releasing interferon γ andcolony stimulating factors that in turn activate phagocytic leukocytes.The activated phagocytic leukocytes are induced to express a number ofspecific cells surface molecules termed homing receptors, which serve toattach the phagocytes to target endothelial cells. Inflammatoryresponses can be reduced or eliminated by treatment with the therapeuticagents of the present invention. For example, antibodies against ACT-4or ACT-4-L block activation of, or eliminate activated, CD4⁺ cells,thereby preventing these cells from releasing molecules required foractivation of phagocytic cell types.

[0161] e. Infectious Agents

[0162] The invention also provides methods of augmenting the efficacy ofvaccines in preventing or treating diseases and conditions resultingfrom infectious agents. Therapeutic agents having the capacity toactivate CD4⁺ T-cells and/or B-cells (e.g., certain monoclonalantibodies against an ACT-4 or ACT-4-L ligand) are administered shortlybefore, concurrently with, or shortly after the vaccine containing aselected antigen. The therapeutic agent serves to augment the immuneresponse against the selected antigen. These methods may be particularlyadvantageous in patients suffering from immune deficiency diseases.

[0163] f. HTLV-I Infections

[0164] Antibodies against ACT-4-h-L-1 are also useful for killingHTLV-I-infected cells. As noted above, such cells express a gp34 antigenthat is identical or nearly identical to ACT-4-h-1. These methods areusually performed in vivo or ex vivo on HTLV-I-infected individuals.However, the methods are also effective for killing HTLV-I-infected HIVcells in vitro. For example, the methods are particularly useful forprotecting hospital workers from infection through contact with tissuesamples under analysis. The risk of HTLV-I infection can be reduced bytreating the samples according to the present methods (provided ofcourse that the samples are being tested for something other than thepresence of HTLV-I).

[0165] Antibodies against ACT-4-h-L-1 and also antibodies againstACT-4-h-1 may be effective to reduce or eliminate perturbations of theimmune system that have been observed in individuals suffering fromHTLV-I infection. Such perturbations may result from the presence ofACT-4-h-L-1 as a surface antigen on HTLV-I infected T-cells, which wouldallow the infected T-cells to interact with CD4⁺ T-cells via an ACT-4receptor.

[0166] g. Treatment of AIDS

[0167] HIV virus is known to infect human CD4⁺ T-cells by binding to theCD4 receptor. However, it is likely that for productive infection tooccur the CD4 receptor must interact with another receptor present onthe receptor of CD4⁺ T-cells. The identification of ACT-4 as a receptoron the surface of activated T-cells suggests that ACT-4 and/or itsligand ACT-4-L may interact with CD4 and contribute to HIV infection ofT-cells. If so, therapeutically effective amounts of therapeutic agentstargeted against ACT-4 or ACT-4-L may be effective in aborting HIVinfection and thereby treating AIDS. These therapeutic agents may alsobe effective for killing HIV-infected CD4⁺ T-cells either in vivo or invitro. In vitro methods have utility in e.g. protecting hospital workersfrom accidental infection as discussed above.

[0168] The following examples are offered to illustrate, but not tolimit, the invention.

EXAMPLES Example 1 A Monoclonal Antibody Against ACT-4-h-1

[0169] Mice were immunized with PHA-transformed T-lymphoblasts.Splenocytes from immunized mice were fused with SP2/O myeloma cells andhybridomas secreting antibodies specific for the T-cell clone wereselected. The hybridomas were cloned by limiting dilution. A monoclonalantibody, designated L106, produced by one of the resulting hybridoma,was selected for further characterization. The L106 antibody was foundto have an IgG1 isotype. A hybridoma producing the antibody, designatedHBL106 has been deposited as ATCC HB11483.

Example 2 Cellular Distribution of Polypeptide Recognized by L106Antibody

[0170] Samples containing the antibody L106 were made available tocertain participants at the Fourth International Workshop and Conferenceon Human Leucocyte Differentiation Antigens (Vienna 1989) for thepurpose of identifying tissue and cell types which bind to the L106antibody. The data from the workshop are presented in Leukocyte TypingIV (ed. W, Knapp, Oxford U. Press, 1989) (incorporated by reference forall purposes) and an accompanying computer data base available fromWalter R. Gilks, MRC Biostatistics Unit, Cambridge University, England.This reference reports the L106 antibody binds a polypeptide of about 50kDa. This polypeptide was reported to be present on HUT-102 cells (atransformed T-cell line), PHA-activated peripheral blood lymphocytes, anEBV-transformed B-lymphoid cell line, and HTLV-II transformed T-cellline, PMA-activated tonsil cells, ConA- or PHA-activated PBLs, andPMA-activated monocytes. The polypeptide was reported to besubstantially absent on inter alia resting basophils, endothelial cells,fibroblasts, interferon γ-activated monocytes, peripheral non-T-cells,peripheral granulocytes, peripheral monocytes, peripheral mononuclearcells, peripheral T cells, and peripheral red blood cells.

[0171] The present inventors have obtained data indicating that the 50kDa polypeptide (hereinafter “ACT-4-h-1 receptor”) is preferentiallyexpressed on the CD4+subspecies of activated T-cells. In one series ofexperiments, cell-specific ACT-4-h-1 expression was analyzed onunfractionated PBLs by a two-color staining method. PBL were activatedwith PHA for about two days (using the culture conditions described inExample 3), and analyzed for cell-surface expression of ACT-4-h-1 ondifferent cellular subtypes by staining with two differently-labelledantibodies (FITC and PE labels). Labels were detected by FACS™ analysisessentially as described by Picker et al., J. Immunol. 150:1105-1121(1993) (incorporated by reference for all purposes). One antibody, L106,was specific for ACT-4-h-1, the other antibody was specific for aparticular leukocyte subtype. FIG. 1 shows three charts in which L106staining is shown on the Y-axis of each chart, and anti-CD4, anti-CD8and anti-CD19 staining as the X-axes of the respective charts. For thechart stained with anti-CD4, many cells appear as double positives(i.e., express both CD4 and ACT-4-h-1). For the chart stained withanti-CD8, far fewer cells appear as double positives. For the chartstained with anti-CD19 (a B-cell marker), double-positive cells aresubstantially absent.

[0172] In another series of experiments expression of ACT-4-h-1 wasanalyzed by single-color staining on isolated cell types. Cells werestained with fluorescently labelled L106 antibody and the label wasdetected by FACS™ analysis. See Engleman et al., J. Immunol.127:2124-2129 (1981) (incorporated by reference for all purposes). Insome experiments, cells were activated by PHA stimulation for about twodays (again using the culture conditions described in Example 3). Theresults from this experiment, together with those from the two-colorstaining experiment described supra, are summarized in Table 1. Table 1shows that about 80% of activated CD4⁺ cells expressed ACT-4-h-1 with amean channel fluorescence of >20, irrespective whether the CD4⁺ cellsare isolated (one-color staining) or in unfractionated PBLs (two-colorstaining). The level of expression of ACT-4-h-1 on activated CD8⁺ cellsis much lower than on activated CD4⁺ T-cells in the two-color stainingexperiment, and very much lower in the one-color staining. Thus, theextent of expression on activated CD8⁺ cells appears to depend onwhether the CD8⁺ cells are fractionated from other PBLs beforeactivation. In unfractionated CD8⁺ cells (two-color staining), about 10%of cells express ACT-4-h-1, with a mean channel fluorescence of about 4.In the fractionated cells, only about 4% of cells express ACT-4-h-1 witha mean channel fluorescence of about 2. These data suggest thatACT-4-h-1 is expressed only on a small subtype of activated CD8⁺ cellsand that this subtype is somewhat more prevalent when the CD8+cells areactivated in the presence of other PBLs.

[0173] Table 1 also indicates that ACT-4-h-1 was substantially absent onall resting leukocyte subtypes tested (i.e., CD4⁺ T-cells, CD8⁺ T-cells,CD19⁺ B-cells, CD14⁺ monocytes, granulocytes and platelets), and wasalso substantially absent on activated B-cells and monocytes. ACT-4-h-1was also found to be substantially absent on most tumor cell linestested. However, Molt3, Raji and NC37 cell lines did show a low level ofexpression. TABLE 1 CELL SPECIFICITY OF ACT-4-h-1 EXPRESSION Expressionof ACT-4-h-1 % Cells MCF¹ Two Color Staining CD4⁺ T-Cells (resting) <2<2 CD4⁺ T-Cells (activated)² 80 25 CD8⁺ T-Cells (resting) <2 <2 CD8⁺T-Cells (activated) 10 4 CD19⁺ B-Cells (resting) <2 <2 CD19⁺ B-Cells(activated) <2 <2 CD14⁺ Monocytes (resting) <2 <2 CD14⁺ Monocytes(activated) <2 <2 One Color Staining PBLs (resting) <2 3 PBLs(activated) 50 27 CD4⁺ (resting) <2 <2 CD4⁺ (activated) 80 22 CD8⁺(resting) <2 <2 CD8⁺ (activated) 4 2 Granulocytes <2 <2 Platelets <2 <2Tumor Lines Molt-4, CEM, Hut 78, H9, Jurkat <2 <2 HPB-ALL, Sezary, T-AU<2 <2 Molt-3 20 3 B-LCL, Arent, RML, JY, KHY, PGf <2 <2 MSAB, CESS,9037, 9062 <2 <2 Dandi, Ramos, Namalwa <2 <2 Raji, NC37 30 4 U937,THP-1, HL-60 <2 <2 Kgla, K562, HEL <2 <2

Example 3 Time Course of ACT-4-h-1 Expression Responsive to CD4⁺ T-cellActivation

[0174] CD4⁺ T-cells were tested for expression of ACT-4-h-1 receptors inresponse to various activating stimuli. CD4⁺ T-cells were purified fromperipheral blood mononuclear cells by solid-phase immunoadsorption(“panning”). 5×10⁴ CD4⁺ T-cells were cultured with an activating agentin microtiter wells containing RPMI medium supplemented with 10% humanserum. Three different activating agents were used: (1) 5×10⁴ irradiated(3000 rads) monocytes, (2) PHA (1 μg/ml) and (3) tetanus toxoid (5μg/ml). ³H-thymidine was added to the cultures 12-16 h before harvest.After harvest, cells were tested for the expression of cell surfaceantigens by incubation with various labelled antibodies (L106, anti-CD4and anti-CD8), as described by Engleman et al., J. Immunol.127:2124-2129 (1981).

[0175]FIG. 2 shows the appearance of ACT-4-h-1 in response toalloantigen activation. Before activation, no expression was observed.The percentage of cells expressing the ACT-4-h-1 receptor increases withtime, peaking at about 30% after about seven days of alloantigenactivation. The results also show that essentially all cells expressingACT-4-h-1 also expressed the CD4 receptor and that essentially no suchcells expressed the CD8 receptor. FIG. 3 presents similar data for theappearance of ACT-4-h-1 in response to tetanus toxoid activation. Again,the percentage of cells expressing ACT-4-h-1 peaked at about seven days.However, at this time a higher percentage of cells (about 60%) expressedthe receptor. FIG. 4 presents similar data for the appearance ofACT-4-h-1 on CD4⁺ T-cells in response to PHA activation. In thissituation, the percentage of CD4⁺ T-cells expressing the receptor peaksat about 65% after three days of activation.

[0176] It is concluded that ACT-4-h-1 is a CD4⁺ T-cell activationantigen that is expressed in response to diverse activating stimuli.

Example 4 Cloning ACT-4-h-1 cDNA

[0177] The cDNA clone for the ACT-4-h-1 receptor was isolated using aslightly modified COS cell expression system, first developed by Aruffo& Seed, supra. RNA was isolated from 72-hour PHA activated humanperipheral blood lymphocytes. Total RNA was extracted with TRI-reagent(Molecular Research Center), and poly(A)+ RNA was isolated by oligodT-magnetic bead purification (Promega). cDNA was synthesized by themethod of Gubler & Hoffman, Gene 25:263-369 (1982) using superscriptreverse transcriptase (Gibco/BRL) and an oligo dT primer. The bluntedcDNA was ligated to non-self-complementary BstXl adaptors and passedover a sephacryl S-400 spin column to remove unligated adaptors andsmall fragments (<300 base pairs). The linkered cDNA was then ligatedinto a BstXl cut eukaryotic expression vector, pcDNA-IRL, an ampicillinresistant version of pcDNA-I(Invitrogen). The precipitated and washedproducts of the ligation reaction were electroporated into E. colistrain WM1100 (BioRad). Plating and counting of an aliquot of thetransformed bacteria revealed a total count of 2 million independentclones in the unamplified library. Average insert size was determined tobe 1.2 kb. The bulk of the library was amplified in liquid culture, 250ml standard LB media. Plasmid was recovered by alkaline lysis andpurified over an ion-exchange column (Qiagen).

[0178] Sub-confluent COS-7 cells were transfected with the purifiedplasmid DNA by electroporation. Cells were plated on 100 mm dishes andallowed to grow for 48 hours. Cells were recovered from the plates withPBS-EDTA solution, incubated with monoclonal antibody L106, and werepanned according to standard procedures. A second round panning revealedenrichment as numerous COS cells adsorbed to the plates. Episomal DNAwas recovered from the immunoselected cells by the Hirt method, andelectroporated into bacteria for amplification.

[0179] Bacteria transformed with plasmid from the second round Hirtpreparation were diluted into small pools of about 100 colonies. Thepools were amplified and their DNA purified and tested for the abilityto confer expression of the L106 antigen on COS-7 cells byimmunofluorescence. Phycoerythrin-conjugated L106 antibody was used tostain COS-7 cell monolayers and the cells were then examined by manualimmunofluorescence microscopy. Miniprep DNA from four out of eight poolswas positive when tested for expression. The pool with the bestexpression, pool E, was divided into smaller pools of 12 colonies. Threeout of eight sub-pools were positive, and sub-pool E1 was plated toallow for the analysis of single colonies. Clone E1-27 was found toconfer high level expression of ACT-4-h-1 receptor on the surface oftransfected COS cells.

Example 5 cDNA Sequence Analysis

[0180] The insert from the clone designated E1-27.was subcloned intopBluescript and sequenced by the dideoxy chain termination method, usingthe T7 polymerase autoread sequencing kit (Pharmacia) on an ALFsequencer (Pharmacia). Restriction mapping revealed several convenientsites for subcloning. Five subclones were generated in pBluescript andwere sequenced on both strands with M13 forward and universal primers.

[0181] The cDNA and deduced amino acid sequences of ACT-4-h-1 are shownin FIG. 5. The ACT-4-h-1 cDNA sequence of 1,137 base pairs contains a14-bp 5′ untranslated region and a 209-bp 3′ untranslated region. AnAATAAA polyadenylation signal is present at position 1,041 followed byan 80-bp poly A tail starting at position 1,057. The longest openreading frame begins with the first ATG at position 15 and ends with aTGA at position 846. The predicted amino acid sequence is that of atypical type 1 integral membrane protein. Hydrophobicity analysisrevealed a putative signal sequence following the initiating ATG, with ashort stretch of basic residues followed by a longer stretch ofhydrophobic residues. A predicted signal peptide cleavage site ispresent at residue 22 or 24 (the latter being the more likely by thecriteria of von Heijne, Nucleic Acids Res. 14:4683-4690 (1986))(incorporated by reference for all purposes), leaving a mature proteinof 253 amino acid residues (or 255 amino acids, if cleavage occurs atthe less probable site). Hydrophobicity analysis also reveals a singlelarge stretch of 27 hydrophobic residues predicted to be thetransmembrane domain, which predicts an extracellular domain of 189 (or191) amino acids and an intracellular domain of 37 amino acids. Theextracellular domain is cysteine rich, where 18 cysteines are foundwithin a stretch of 135 amino acids. The predicted molecular mass (Mr)for the mature protein is 27,400, and there are two potentialN-glycosylation sites at amino acid residues 146 and 160.

[0182] Comparison of the amino acid sequence of ACT-4-h-1 with knownsequences in the swiss-prot database using the BLAZE program reveals asequence similarity with members of the nerve growth factor receptorsuperfamily. Amino acid sequences are at least 20% identical for NGF-R,TNF-R, CD40, 41-BB, and fas/APO-1,. and 62% for OX-40, allowing for gapsand deletions. Alignments of the various proteins reveal theconservation of multiple cysteine rich motifs. Three of these motifs arepresent in ACT-4-h-1 and OX-40, compared with four such motifs in NGF-Rand CD40.

[0183] Comparison of the nucleotide sequence of ACT-4-h-1 with knownsequences in the Genbank and EMBL databases using the programs BLAST andFASTDB revealed a high degree of sequence similarity with only onemember of the nerve growth factor receptor family, OX-40. Allowing forgaps and insertions, the sequence identity is 66%. Comparison of theACT-4-h-1 and OX-40 nucleotide sequences reveals that both contain a14-bp 5′ untranslated region, and both contain approximately 80-bp polyA tails. In ACT-4-h-1, however, there is a slight lengthening of the 3′untranslated region from 187-bp to 209-bp, and there is a lengthening ofthe coding region from 816-bp to 834-bp, a difference of 18-bp or 6amino acid insertions. Aligning the two amino acid sequences revealsthat four of the amino acid insertions occur prior to the signalsequence cleavage site. Thus, the mature ACT-4-h-1 receptor proteincontains one more amino acid residue than OX-40 (i.e., 253 vs. 252 aminoacids). Remarkably, the ACT-4-h-1 nucleotide sequence is much more GCrich, than the OX-40 sequence (70% v. 55%) indicating that the twosequences will not hybridize under stringent conditions.

Example 6 Production of Stable ACT-4-h-1 Transfectants

[0184] An XbaI-HindIII fragment was excised from the construct describedin Example 4, and inserted into XbaI/HindIII-digested pcDNA-I-neo(Invitrogen) to generate an expression vector termed ACT-4-h-1-neo (FIG.6). This vector was linearized with Sf1 and electroporated into threeeukaryotic cell lines. These cell lines were SP2/O (a mouse myelomaderived from the Balb/c strain), Jurkat (a transformed human T-cellline) and COS-7 (an adherent monkey cell line). After a 48-h recoveryperiod, transformed cells were selected in 1 mg/ml G418 (Gibco). Afterthree weeks of selection, neo-resistant cell lines were incubated with asaturating concentration of L106 antibody, washed and overlayered onto100 mm petri dishes coated with goat anti-mouse IgG to select for cellsexpressing ACT-4-h-1. After washing off unbound cells, adherent cellswere recovered and expanded in tissue culture. Cell lines were subjectto two further rounds of panning and expression. The resulting celllines were shown by direct immunofluorescence staining to expressabundant ACT-4-h-1 (FIG. 7).

[0185] The same strategy and principles are used to obtain a stable cellline expressing ACT-4-L-h-1 (See Example 10).

Example 7 Production of an ACT-4-h-1-Immunoglobulin Fusion Protein

[0186] A soluble fusion protein has been constructed in which theextracellular domain of ACT-4-h-1 is linked via its C-terminal to theN-terminal of the constant domain of a human immunoglobulin. The vectorencoding ACT-4-h-1 described in Example 4 was cleaved with SmaI and NotIto excise all ACT-4-h-1 sequences downstream of the SmaI site includingthe transmembrane, cytoplasmic and 3′ untranslated regions. Theremaining region encodes the soluble extracellular portion of ACT-4-h-1(FIG. 8). The source of the immunoglobulin constant region to be joinedto the ACT-4-h-1 extracellular domain was a plasmid termed 5K-41BB-Eg1(Proc. Natl. Acad. Sci. (USA) 89: 10360-10364) (incorporated byreference for all purposes). This plasmid contains a 1.3 kb BamHI/EagIgenomic fragment encoding the hinge, CH2 and terminal CH3 domains ofhuman Ig, isotype gamma 1. The fragment required modification forinsertion into the SmaI/NotI ends of the ACT-4-h-1 vector, whilepreserving the peptide reading frame across the SmaI junction to beformed by blunt-end ligation. The vector 5k-41BB-Eg1 was cut with BamH1and the resulting 5′ extensions were filled with Klenow fragment. Thevector was then cut with EagI releasing the 1.3 kb fragment with bluntand NotI compatible ends. This fragment was ligated with SmaI/NotIdigested ACT-4-h-1 vector. The ligation mix was electroporated into E.coli and multiple transformant clones screened with PCR using ACT-4-h-1and IgG1 nucleotide fragments as primers.

[0187] Plasmids containing the ACT-4-h-1-IgG1 coding were electroporatedinto COS cells. The cells were allowed to grow for five days at whichpoint their supernatants were harvested and sterile filtered through a0.2 micron membrane. The supernatants were tested for expression ofACT-4-h-1-IgG1 by dot blotting. Supernatants were blotted ontomitrocellulose and blocked with 5% nonfat dry milk. Replica blots wereprobed with antibody L106 or alkaline phosphatase-labelled goatanti-human immunoglobulin IgG (American Qualex). Antibody L106 wasdetected with an alkaline phosphatase labelled goat anti-mouse IgG.NBT/BCIP (Pierce) was used as a colorimetric substrate. High producingpositive clones were sequenced at the junction site to confirm propervector construction. The resulting fusion gene is depicted in FIG. 9.

Example 8 Identification of Cells Types Expressing a Ligand to ACT-4-h-1

[0188] Cell types expressing a ligand to ACT-4-h-1 were identified byindirect staining combined with flow cytometry using the ACT-4-h-1recombinant immunoglobulin fusion protein described in Example 7(hereinafter ACT-4-h-1-Rg) as a probe. Bound fusion protein was detectedusing phycoerythrin-conjugated anti-human Ig. These experiments revealedthat a ligand to ACT-4-h-1 was expressed at low levels on a few B-cellslines, including a Burkitt lymphoma cell line (Jiyoye), and theEBV-transformed LCL's 9037, 9059, and MSAB. These cell lines scored 5%positive with a MCF of 3. See Table 2. With the cell line Jiyoye, it waspossible to enrich for cells staining positive by the panning procedureto yield a cell line Jo-P5, which stained 90% positive with a MCF of 10.Other cell lines tested, including PBMC's and purified subpopulations offreshly isolated T-cells, B-cells, monocytes, dendritic cells, mostT-cell tumor lines and myelomonocytic tumor cell lines, weresubstantially absent of a ligand to ACT-4-h-1. However, two HTLV-Iinfected T cell lines, HUT-102 and MT-2 expressed ACT-4-L-h-1, thelatter at extremely high levels.

[0189] The experiment was repeated after activation of cells usingeither PHA or a combination of PMA/ionomycin. PBMC's activated with 2μg/ml of PHA for three days stained between 2-10% positive for a ligandto ACT-4-h-1, depending on the donor. Activation of B-LCL cells andBurkit lymphoma lines using a combination of PMA (10 ng/ml) andionomycin (500 ng/ml) induced substantial expression of ligand for somecell lines, particularly those such as MSAB that showed low levels ofexpression in the resting state. See Table 2. Unfractionated B cellsalso showed preferential expression of ligand (15% cells positive,MHC=5). Time course studies on MSAB cells indicated that ligandexpression begins on day 2 following activation, and peaks on day 3 or5. PMA/ionomycin-activation also induced preferential expression ofligand in erythroleukemia cell lines and in one of the threemyelomonocytic cell lines tested, THP-1. PMA/ionomycin activation alsoinduced a low level of expression of ligand in the T-cell lines but notin the other T-cell lines tested. TABLE 2 Expression of Ligand toACT-4-h-1 on Different Cell Types TUMOR CELL LINE SCREEN RestingActivated¹

MSAB 2-5%² mcf³ 4 80% mcf 21 CESS <2 70% mcf 25 JY <2 40% 9 REM <2 40%11 9059 2-5% mcf 4 40% 6 SKF <2 30% 3 9037 2-5% mcf 4 25% 5 9062 <2 10%5 PGF <2 6% 10 ARENT <2 4% 5 KHY <2 3% 5

Jiyoye 20% mcf 4 7% 5 Daudi <2 10% 5 Naralwa <2 5% 5 Raji <2 <2

(Pre B) NC-37 <2 <2 (B-All) SB <2 15% 5

HSB-2 <2 6% 3 Jurkat <2 <2 Mol +4 <2 <2 Mol +3 <2 <2 HPB-ALL <2 <2 HU+78 <2 <2 H9 <2 <2 VB <2 <2

THP-1 <2 25% mcf 5 V937 <2 <2 HL60 <2 <2

HEL <2 25% 5 K562 2% 25% 5

HUT-102 30% mcf 4 MT-2 100% 100

Example 9 Cloning cDNA Encoding an ACT-4-h-1 Ligand

[0190] The cDNA clone for the ligand was isolated using a slightlymodified COS cell expression system, first developed by Aruffo & Seed,supra. RNA was isolated from 72-hour PMA/ionomycin-activated humanEBY-transformed B cells (cell line MSAB). Total RNA was extracted withTRI-reagent (Molecular Research Center), and poly(A)+ RNA was isolatedby oligo dT-magnetic bead purification (Promega). cDNA was synthesizedby the method of Gubler & Hoffman, Gene 25:263-369 (1982) usingsuperscript reverse transcriptase (Gibco/BRL) and an oligo dT primer.The blunted cDNA was ligated to non-self-complementary BstXl adaptorsand passed over a Sephacryl S-500 column to remove unligated adaptorsand small fragments (<300 base pairs). The Tinkered cDNA was thenligated into a BstXl cut eukaryotic expression vector, pcDNA(Invitrogen). The ligation products were precipitated, washed andelectroporated into E. coli strain MC1061/P3 generating an unamplifiedlibrary of 100 million independent clones. The average insert size inthe library was 1 kb. The bulk of the library was amplified in 250 mlstandard LB media. Plasmid DNA was recovered by alkaline lysis andpurified over an ion-exchange column (Qiagen).

[0191] Sub-confluent COS-7 cells were transfected with the purifiedplasmid DNA by electroporation. Cells were plated on 100 mm dishes andallowed to grow for 48 hours. Cells were recovered from the plates withPBS-EDTA solution, incubated with monoclonal antibody ACT-4-h-1-Rg, andpanned according to standard procedures. A second round panning numerousCOS cells adsorbed to the plates showing enrichment for cells expressingligand. Episomal DNA was recovered from the immunoselected cells by theHirt method, and electroporated into bacteria for amplification.

[0192] Bacteria transformed with plasmid from the second round selectionwere cloned and amplified. DNA from individual clones was purified andtested for the ability to confer expression of a ligand to ACT-4-h-1 inCOS-7 cells. Phycoerythrin-conjugated ACT-4-h-1-Rg was used to stainCOS-7 cell monolayers and the cells were then examined by manualimmunofluorescence microscopy. Clones #2, 26, and 30 gave high levelexpression of ACT-4-h-1-Rg binding activity.

Example 10 Sequence Analysis of a Ligand to ACT-4-h-1

[0193] The insert from clone 26 in Example 9 was subcloned intopBluescript at the HindIII and XbaI cloning sites. The clone wassequenced by the dideoxy chain termination method, using a T7polymerase-based autoread sequencing kit (Pharmacia) on an ALF sequencer(Pharmacia). Three subclones were generated in pBluescript and weresequenced on both strands with M13 forward and universal primers. ThecDNA and predicted amino acid sequences of clone 26 are shown in FIG.10. The polypeptide formed by the predicted amino acid sequence isdesignated ACT-4-L-h-1.

[0194] The ACT-4-L-h-1 cDNA sequence contains 1079 base pairs, with a137 bp 5′ UTR and a 379 bp 3′ UTR. An AATAAA polyadenylation signal ispresent at position 1024 followed by a 20 base poly A tail beginning atposition 1049. Sequence analysis reveals a single open reading framewhich encodes a 183 amino acid polypeptide, with a calculated molecularweight of 21,000. The open reading frame begins with the first ATG atposition 149 and ends with a TGA at position 698. The ATG is flankedwith a Kozak consensus initiation sequence with an A position at −3 anda G at +4. Hydrophobicity analysis reveals that the predicted amino acidsequence is that of a type II membrane protein with a singletransmembrane domain of approximately 27 aa in the amino terminalportion of the protein. Also there are four N-linked glycosylation sitesin the C-terminal portion of the molecule.

[0195] Comparison of the nucleotide sequence of ACT-4-L-h-1 with knownsequences in the Genbank and EMBL databases reveals no significanthomology to known genes except for a gene encoding a protein designatedgp34 by Miura et al., Mol. Cell Biol. 11:1313-1325 (1991). The cDNAsequence of the coding region of the ACT-4-L-h-1 ligand is identical tothat of gp34. However, the ACT-4-L-h-1 cDNA contained an additional 112nucleotides at the 5′ end compared with the gp34 sequence. Because theadditional nucleotides occur within the 5′ untranslated region, theirpresence is unlikely to alter the expression product. Most likely, theACT-4-L-h-1 clone was derived from a more complete reverse transcriptand is more representative of the in vivo 5′ end. Possibly the extrasequence could be involved in regulating the translation of the protein.

[0196] Comparison of the predicted amino acid sequence of theACT-4-L-h-1 ligand with known sequences in the Protein InformationResource (PIR) database using the FastDB program revealed an identitywith gp34, and a very weak homology with TNF alpha. A secondarystructure prediction algorithm developed by Chou & Fasman predicts thatthe ACT-4-L-h-1 ligand and TNF-alpha are both likely to form significantamounts of beta structures. This prediction is consistent with theobservation that other members of the TNF family of proteins are allconformed or predicted to form beta jelly roll configurations rich inbeta structures.

[0197] For the purposes of clarity and understanding, the invention hasbeen described in these examples and the above disclosure in somedetail. It will be apparent, however, that certain changes andmodifications may be practiced within the scope of the appended claims.All publications and patent applications are hereby incorporated byreference in their entirety for all purposes to the same extent as ifeach were so individually denoted.

1 4 1 1058 DNA Homo sapiens CDS (15)..(845) 1 cagcagagac gagg atg tgcgtg ggg gct cgg cgg ctg ggc cgc ggg ccg 50 Met Cys Val Gly Ala Arg ArgLeu Gly Arg Gly Pro 1 5 10 tgt gcg gct ctg ctc ctc ctg ggc ctg ggg ctgagc acc gtg acg ggg 98 Cys Ala Ala Leu Leu Leu Leu Gly Leu Gly Leu SerThr Val Thr Gly 15 20 25 ctc cac tgt gtc ggg gac acc tac ccc agc aac gaccgg tgc tgc cac 146 Leu His Cys Val Gly Asp Thr Tyr Pro Ser Asn Asp ArgCys Cys His 30 35 40 gag tgc agg cca ggc aac ggg atg gtg agc cgc tgc agccgc tcc cag 194 Glu Cys Arg Pro Gly Asn Gly Met Val Ser Arg Cys Ser ArgSer Gln 45 50 55 60 aac acg gtg tgc cgt ccg tgc ggg ccg ggc ttc tac aacgac gtg gtc 242 Asn Thr Val Cys Arg Pro Cys Gly Pro Gly Phe Tyr Asn AspVal Val 65 70 75 agc tcc aag ccg tgc aag ccc tgc acg tgg tgt aac ctc agaagt ggg 290 Ser Ser Lys Pro Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg SerGly 80 85 90 agt gag cgg aag cag ctg tgc acg gcc aca cag gac aca gtc tgccgc 338 Ser Glu Arg Lys Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg95 100 105 tgc cgg gcg ggc acc cag ccc ctg gac agc tac aag cct gga gttgac 386 Cys Arg Ala Gly Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp110 115 120 tgt gcc ccc tgc cct cca ggg cac ttc ttc cca ggc gac aac caggcc 434 Cys Ala Pro Cys Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala125 130 135 140 tgc aag ccc tgg acc aac tgc acc ttg gct ggg aag cac accctg cag 482 Cys Lys Pro Trp Thr Asn Cys Thr Leu Ala Gly Lys His Thr LeuGln 145 150 155 ccg gcc agc aat agc tcg gac gca atc tgt gag gac agg gacccc cca 530 Pro Ala Ser Asn Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp ProPro 160 165 170 2 277 PRT Homo sapiens deduced amino acid sequence ofACT-4-h-1 2 Met Cys Val Gly Ala Arg Arg Leu Gly Arg Gly Pro Cys Ala AlaLeu 1 5 10 15 Leu Leu Leu Gly Leu Gly Leu Ser Thr Val Thr Gly Leu HisCys Val 20 25 30 Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His Glu CysArg Pro 35 40 45 Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln Asn ThrVal Cys 50 55 60 Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val Ser SerLys Pro 65 70 75 80 Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly SerGlu Arg Lys 85 90 95 Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg CysArg Ala Gly 100 105 110 Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val AspCys Ala Pro Cys 115 120 125 Pro Pro Gly His Phe Ser Pro Gly Asp Asn GlnAla Cys Lys Pro Trp 130 135 140 Thr Asn Cys Thr Leu Ala Gly Lys His ThrLeu Gln Pro Ala Ser Asn 145 150 155 160 Ser Ser Asp Ala Ile Cys Glu AspArg Asp Pro Pro Ala Thr Gln Pro 165 170 175 Gln Glu Thr Gln Gly Pro ProAla Arg Pro Ile Thr Val Gln Pro Thr 180 185 190 Glu Ala Trp Pro Arg ThrSer Gln Gly Pro Ser Thr Arg Pro Val Glu 195 200 205 Val Pro Gly Gly ArgAla Val Ala Ala Ile Leu Gly Leu Gly Leu Val 210 215 220 Leu Gly Leu LeuGly Pro Leu Ala Ile Leu Leu Ala Leu Tyr Leu Leu 225 230 235 240 Arg ArgAsp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly 245 250 255 GlySer Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser 260 265 270Thr Leu Ala Lys Ile 275 3 1048 DNA Homo sapiens CDS (138)..(686) 3ggccctggga cctttgccta ttttctgatt gataggcttt gttttgtctt tacctccttc 60tttctgggga aaacttcagt tttatcgcac gttccccttt tccatatctt catcttccct 120ctacccagat tgtgaag atg gaa agg gtc caa ccc ctg gaa gag aat gtg 170 MetGlu Arg Val Gln Pro Leu Glu Glu Asn Val 1 5 10 gga aat gca gcc agg ccaaga ttc gag agg aac aag cta ttg ctg gtg 218 Gly Asn Ala Ala Arg Pro ArgPhe Glu Arg Asn Lys Leu Leu Leu Val 15 20 25 gcc tct gta att cag gga ctgggg ctg ctc ctg tgc ttc acc tac atc 266 Ala Ser Val Ile Gln Gly Leu GlyLeu Leu Leu Cys Phe Thr Tyr Ile 30 35 40 tgc ctg cac ttc tct gct ctt caggta tca cat cgg tat cct cga att 314 Cys Leu His Phe Ser Ala Leu Gln ValSer His Arg Tyr Pro Arg Ile 45 50 55 caa agt atc aaa gta caa ttt acc gaatat aag aag gag aaa ggt ttc 362 Gln Ser Ile Lys Val Gln Phe Thr Glu TyrLys Lys Glu Lys Gly Phe 60 65 70 75 atc ctc act tcc caa aag gag gat gaaatc atg aag gtg cag aac aac 410 Ile Leu Thr Ser Gln Lys Glu Asp Glu IleMet Lys Val Gln Asn Asn 80 85 90 tca gtc atc atc aac tgt gat ggg ttc tatctc atc tcc ctg aag ggc 458 Ser Val Ile Ile Asn Cys Asp Gly Phe Tyr LeuIle Ser Leu Lys Gly 95 100 105 tac ttc tcc cag gaa gtc aac att agc cttcat tac cag aag gat gag 506 Tyr Phe Ser Gln Glu Val Asn Ile Ser Leu HisTyr Gln Lys Asp Glu 110 115 120 gag ccc ctc ttc caa ctg aag aag gtc aggtct gtc aac tcc ttg atg 554 Glu Pro Leu Phe Gln Leu Lys Lys Val Arg SerVal Asn Ser Leu Met 125 130 135 gtg gcc tct ctg act tac aaa gac aaa gtctac ttg aat gtg acc act 602 Val Ala Ser Leu Thr Tyr Lys Asp Lys Val TyrLeu Asn Val Thr Thr 140 145 150 155 gac aat acc tcc ctg gat gac ttc catgtg aat ggc gga gaa ctg att 650 Asp Asn Thr Ser Leu Asp Asp Phe His ValAsn Gly Gly Glu Leu Ile 160 165 170 ctt atc cat caa aat cct ggt gaa ttctgt gtc ctt tgaggggctg 696 Leu Ile His Gln Asn Pro Gly Glu Phe Cys ValLeu 175 180 atggcaatat ctaaaaccag gcaccagcat gaacaccaag ctgggggtggacagggcatg 756 gattcttcat tgcaagtgaa ggagccaccc agctcagcca cgtgggatgtgacaagaagc 816 agatcctggc cctcccgccc ccacccctca gggatattta aaacttattttatataccag 876 ttaatcttat ttatccttat attttctaaa ttgcctagcc gtcacaccccaagattgcct 936 tgagcctact aggcaccttt gtgagaaaga aaaaatagat gcctcttcttcaagatgcat 996 tgtttctatt ggtcaggcaa ttgtcataat aaacttatgt cattgaaaac gg1048 4 183 PRT Homo sapiens predicted amino acid sequence of ACT-4-L-h-14 Met Glu Arg Val Gln Pro Leu Glu Glu Asn Val Gly Asn Ala Ala Arg 1 5 1015 Pro Arg Phe Glu Arg Asn Lys Leu Leu Leu Val Ala Ser Val Ile Gln 20 2530 Gly Leu Gly Leu Leu Leu Cys Phe Thr Tyr Ile Cys Leu His Phe Ser 35 4045 Ala Leu Gln Val Ser His Arg Tyr Pro Arg Ile Gln Ser Ile Lys Val 50 5560 Gln Phe Thr Glu Tyr Lys Lys Glu Lys Gly Phe Ile Leu Thr Ser Gln 65 7075 80 Lys Glu Asp Glu Ile Met Lys Val Gln Asn Asn Ser Val Ile Ile Asn 8590 95 Cys Asp Gly Phe Tyr Leu Ile Ser Leu Lys Gly Tyr Phe Ser Gln Glu100 105 110 Val Asn Ile Ser Leu His Tyr Gln Lys Asp Glu Glu Pro Leu PheGln 115 120 125 Leu Lys Lys Val Arg Ser Val Asn Ser Leu Met Val Ala SerLeu Thr 130 135 140 Tyr Lys Asp Lys Val Tyr Leu Asn Val Thr Thr Asp AsnThr Ser Leu 145 150 155 160 Asp Asp Phe His Val Asn Gly Gly Glu Leu IleLeu Ile His Gln Asn 165 170 175 Pro Gly Glu Phe Cys Val Leu 180

What is claimed is:
 1. A purified ACT-4-L ligand polypeptide having asegment between 5 and 160 contiguous amino acids from an amino acidsequence shown in FIG.
 10. 2. The polypeptide of claim 1 that exhibitsat least eighty percent sequence identity to the amino acid sequence ofFIG.
 10. 3. The polypeptide of claim 2 having an antigenic determinantcommon to a protein with the amino acid sequence shown in FIG.
 10. 4.The polypeptide of claim 3 comprising an intracellular domain, atransmembrane domain, or an extracellular domain.
 5. The polypeptide ofclaim 1 that comprises an extracellular domain of an ACT-4-L ligand. 6.The polypeptide of claim 1 that is glycosylated.
 7. The polypeptide ofclaim 6 further comprising a linked second polypeptide.
 8. Thepolypeptide of claim 7, wherein the second polypeptide is animmunoglobulin constant domain.
 9. A purified extracellular domain of anACT-4-L ligand comprising at least five contiguous amino acids from thefull-length ACT-4-L-h-1 extracellular domain.
 10. The extracellulardomain of claim 9 that is full-length.
 11. The extracellular domain ofclaim 9 that is in soluble form.
 12. The extracellular domain of claim11 that specifically binds to the ACT-4-L-h-1 ligand.
 13. Theextracellular domain of claim 11 that specifically binds to theACT-4-h-1 receptor.
 14. The extracellular domain of claim 13 consistingessentially of a domain that specifically binds to the ACT-4-h-1receptor.
 15. The extracellular domain of claim 9 that is labelled. 16.The extracellular domain of claim 9 that inhibits in vitro activation ofCD4⁺ T cells expressing the ACT-4-h-1 receptor on their surface.
 17. Theextracellular domain of claim 9 that stimulates in vitro activation ofCD4⁺ T cells expressing the ACT-4-h-1 receptor on their surface.
 18. Theextracellular domain of claim 9 that competes with an antibodydesignated L106 for specific binding to the ACT-4-h-1 receptor.
 19. Theextracellular domain of claim 9 that competes with the ACT-4-L-h-1ligand for binding to an antibody.
 20. The extracellular domain of claim9 further comprising a linked second polypeptide.
 21. The extracellulardomain of claim 20, wherein the second polypeptide is a constant regionof an immunoglobulin heavy chain.
 22. An ACT-4 receptor polypeptideconsisting essentially of a domain that specifically binds to theACT-4-L-h-1 ligand.
 23. A humanized monoclonal antibody comprising ahumanized heavy chain and a humanized light chain: (1) the humanizedlight chain comprising three complementarity determining regions (CDR1,CDR2 and CDR3) having amino acid sequences from the correspondingcomplementarity determining regions of a light chain of a nonhumanantibody that specifically binds to an extracellular domain of theACT-4-L-h-1 ligand, and having a variable region framework sequencesubstantially identical to a human light chain variable region frameworksequence; and (2) the humanized heavy chain comprising threecomplementarity determining regions (CDR1, CDR2 and CDR3) having aminoacid sequences from the corresponding complementarity determiningregions a heavy chain of the nonhuman antibody, and having a variableregion framework sequence substantially identical to a human heavy chainvariable region framework sequence; wherein the humanized antibodyspecifically binds to the extracellular domain of the ACT-4-L-h-1 ligandwith a binding affinity having a lower limit of 10⁷ M⁻¹ and an upperlimit that is within five-fold of the binding affinity of the nonhumanantibody.
 24. The humanized monoclonal antibody of claim 23, wherein theACT-4-L-h-1 ligand is on the surface of a B cell and the binding of theantibody to the ligand inhibits activation of the B cell.
 25. Thehumanized monoclonal antibody of claim 23, wherein the ACT-4-L-h-1ligand is on the surface of a B cell and the binding of the antibody tothe ligand stimulates activation of the B-cell.
 26. The humanizedmonoclonal antibody of claim 23, wherein the ACT-4-L-h-1 ligand is onthe surface of a B cell and the binding of the antibody to the ligandinhibits the capacity of the B-cells to activate CD4⁺ T-cells.
 27. Ahuman monoclonal antibody that specifically binds to an extracellulardomain of the ACT-4-L-h-1 ligand.
 28. A purified nucleic acid segmentencoding an ACT-4-L ligand polypeptide of claim
 1. 29. The purifiednucleic acid segment of claim 28 further comprising at least 25contiguous nucleotides between nucleotides 1 and 112 of the5′-untranslated region of the sequence shown in FIG.
 10. 30. A purifiednucleic acid segment having between 15 and 500 contiguous nucleotidesfrom the sequence shown in FIG.
 10. 31. An isolated population of cellssubstantially enriched in B-cells expressing an ACT-4-L ligand on theirsurface.
 32. An isolated stable cell line expressing an ACT-4-L ligandon its surface.
 33. A pharmaceutical composition comprising apharmaceutically active carrier and an agent that specifically binds toan extracellular domain of the ACT-4-L-h-1 ligand.
 34. A method ofsuppressing an immune response in a patient having an immune disease orcondition, the method comprising administering an effective amount of apharmaceutical composition comprising a pharmaceutically active carrierand an agent that specifically binds to the ACT-4-L-h-1 ligand.
 35. Themethod of claim 34, wherein the agent competes with the ACT-4-h-1receptor for specific binding to the ACT-4-L-h-1 ligand.
 36. The methodof claim 35, wherein the agent is a monoclonal antibody.
 37. The methodof claim 36, wherein the monoclonal antibody is humanized.
 38. Themethod of claim 36, wherein the monoclonal antibody is human.
 39. Themethod of claim 35, wherein the agent is an ACT-4-L ligand polypeptide.40. The method of claim 35, wherein the agent is a second ACT-4 receptorpolypeptide consisting essentially of a domain that binds to theACT-4-L-h-1 ligand.
 41. The method of claim 35, wherein theadministering step is performed ex vivo.
 42. A method of suppressing animmune response in a patient having an immune disease or condition, themethod comprising administering an effective amount of a pharmaceuticalcomposition comprising a pharmaceutically active carrier and an agentthat competes with the ACT-4-L ligand for specific binding to theACT-4-h-1 receptor.
 43. The method of claim 42, wherein the agent is amonoclonal antibody.
 44. The method of claim 42, wherein the agent is asecond ACT-4-L ligand polypeptide.
 45. The method of claim 42, whereinthe agent is an ACT-4 receptor polypeptide consisting essentially of adomain that binds to the ACT-4-h-1 receptor.
 46. A method of screeningfor immunosuppressive agents, the method comprising: contacting anACT-4-L-h-1 ligand polypeptide with a potential immunosuppressive agent;and detecting specific binding between the ACT-4-L-h-1 ligandpolypeptide and the agent, the specific binding indicative ofimmunosuppressive activity.
 47. The method of claim 46, wherein theACT-4-L-h-1 ligand polypeptide is immobilized to a solid surface.
 48. Amethod of inducing an immune response to a selected antigen comprising:administering the monoclonal antibody of claim 25 to a patient; andexposing the patient to the selected antigen.
 49. A method of monitoringactivated CD4⁺ T-cells, the method comprising: contacting a tissuesample from a patient with an ACT-4-L ligand polypeptide thatspecifically binds to an extracellular domain of the ACT-4-h-1 receptor;and detecting specific binding between the ACT-4-L ligand polypeptideand the tissue sample to indicate the presence of the activated CD4⁺T-cells.
 50. A method of inhibiting infection of CD4⁺ T-cells by a humanimmune deficiency virus, the method comprising contacting the CD4⁺T-cells with an antibody that specifically binds to an extracellulardomain of the ACT-4 receptor.